The Achilles' Heel of âUltrastableâ Hyperthermophile Proteins

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

The Achilles' heel of 'ultrastable' hyperthermophile proteins : Submillimolar concentrations of SDS stimulate rapid conformational change, aggregation and amyloid formation in proteins carrying overall positive charge Javed Masood Khan, Prerna Sharma, Kanika Arora, Nitin Kishor, Pallavi Kaila, and Purnananda Guptasarma Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01343 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The Achilles' heel of 'ultrastable' hyperthermophile proteins : Sub-millimolar concentrations of SDS stimulate rapid conformational change, aggregation and amyloid formation in proteins carrying overall positive charge

Javed M. Khan#,*, Prerna Sharma, Kanika Arora, Nitin Kishor, Pallavi Kaila and Purnananda Guptasarma*

Centre for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Knowledge City, Sector 81, SAS Nagar, Punjab, India, 140306

* For correspondence: Purnananda Guptasarma, Email: [email protected], Tel : +91-172-2293151, or Javed Masood Khan, Email: [email protected]. # Present Address : Department of Food Science and Nutrition, Faculty of Food and Agricultural Sciences, King Saud University, 2460 Riyadh 11451, Saudi Arabia.

1

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Low concentrations (< 3.0 mM) of the anionic surfactant, SDS, have been shown to induce the formation of amyloid fibers in over twenty different mesophile-derived proteins in the cationic state. It is not known whether SDS has similar effects on hyperthermophile-derived proteins, which are otherwise thought to be ‘ultrastable’ and inordinately resistant to structural perturbations at room temperature. Here, we show that low (< 4.5 mM) concentrations of SDS rapidly induce the formation of aggregates and amyloid fibers in five different ultrastable Pyrococcus furiosus proteins in the cationic state. We also show that amyloid formation is accompanied by the development of a characteristic, negative CD band at ~230 nm. These effects are not seen if the proteins have a net negative charge, or when higher concentrations of SDS are used (which induce helix-formation instead). Our results appear to reveal a potential weakness or ‘Achilles’ heel’ in ultrastable proteins from hyperthermophiles. They also provide very strong support to the view that SDS initially interacts with proteins through electrostatic interactions, and not hydrophobic interactions, eliciting similar effects entirely regardless of protein molecular weight, or structural features such as quaternary structure or tertiary structural stability.

Ultrastable proteins. Thermophile and hyperthermophile microorganisms are known to be able to withstand extreme temperatures, e.g., several archaea exhibit optimal growth at temperatures in the range of 80-100 0C. Proteins with amino acid sequences derived from the proteomes of thermophile and hyperthermophile organisms have been found to retain three-dimensional structure, as well as biological function, at such high temperatures (quite regardless of whether they have been produced in their original hosts, or in heterologous overexpression systems), in 2


Page 2 of 55

Page 3 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

direct contrast to mesophile homologs of such proteins which invariably become unfolded and non-functional1. Various factors are thought to contribute to the stability of hyperthermophilederived ‘ultrastable’ proteins; these include higher levels of hydrophobic interactions, better internal packing of side-chains, and greater numbers of surface salt-bridges2,3, as well as higher contents of proline residues, greater numbers of disulphide bridges, bound metal cofactors, greater

capping of helices and significantly smaller lengths of protein loop sequences4.

Ultrastable proteins also display greater ‘apparent’ stability, in part because their plots of temperature versus difference in free energy (between native and unfolded states) are broader and upshifted5; their kinetic stabilities are also extremely high, i.e., they unfold extremely slowly under conditions which, broadly speaking, are thermodynamically destabilizing and which elicit unfolding, but very slowly. In comparison to mesophiles, hyperthermophile proteins display stronger ion pairing, fewer cavities and higher β-sheet contents. Considering all these differences, hyperthermophile-derived ultrastable proteins constitute a fascinating subject of study, and various approaches such as thermal denaturation, chemical denaturation (use of GdnHCl and urea), thermo-chemical denaturation (combining thermal and chemical denaturation), interactions with co-solvents, lipids or surfactants are being used today, to examine their stabilities in-vitro. Surfactant-induced protein conformational changes. Protein-surfactant and protein-lipid interactions have been studied for a long time. It is well established that surfactants have a 1000times higher ability to unfold proteins in the millimolar concentration range, in comparison to other denaturants like guanidium hydrochloride (GdmHCl) and urea which are only able to effect unfolding at molar concentrations, ostensibly due to poor binding to the protein backbone. Many groups have already reported that amyloid fibrils are formed due to interactions of lipids with 3


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 55

amyloid and non amyloid precursor proteins6,7. Ionic surfactants, especially anionic ones like sodium dodecyl sulphate (SDS), interact with proteins via electrostatic as well as hydrophobic interactions, with the hydrophobic moiety in SDS mimicking the lipids that form an integral part of biological membranes. SDS interacts with many types of proteins, and is capable of unfolding them, with numerous reports suggesting that SDS can also elicit adoption of some non-native structures, primarily of an α-helical nature, with the surfactant inducing helicity8,9. SDS exists in two forms in

solution; at low concentrations it exists as

a monomer, whereas at high

concentrations it forms micelles. The concentration at which SDS forms micelles, which is known as the critical micellar concentration (CMC) is dependent upon ionic strength (salt concentration) and temperature, as well as upon the presence of other solutes, including proteins to which SDS can bind10,11. In addition, there is also a minor dependence on pH, in that SDS undergoes acidification at very low pH (below pH 1.5). It is reported that SDS accelerates the formation of amyloid fibers by the amyloid beta protein and by a few other proteins12, by promoting aggregation of αβ (1-40) and αβ (1-42) only when SDS concentrations are very low, inducing the formation of helical structure at higher SDS concentrations below the denaturationinducing concentration13,14. Thus it may be said that submicellar concentrations of SDS can and do induce profound conformational changes in proteins15. With studies of the interactions of the acyl-coenzyme-A-binding protein with SDS, it is reported that only 1-3 SDS molecules bind to the protein at low SDS concentrations (< 1.3 mM), whereas at higher concentrations as many as ~13 SDS molecules bind per monomer, resulting in changes in secondary as well as tertiary structure16. Another interesting observation relates to the protein, carbonic anhydrase; SDS is reported to fail to unfold the protein when its amino group is acylated, whereas unmodified protein is susceptible to unfolding at very low SDS concentrations17,18. 4


Page 5 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Amyloids and ultrastable proteins. Amyloid fibril formation is associated with several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and type II diabetes mellitus19. Amyloids have special characteristic conformational features, i.e. they are rich in cross β-sheet structures and bind to the dye, thioflavin T (ThT), with a consequent large increase in the fluorescence intensity of ThT. The mechanism of amyloid fibril formation is not yet fully understood. Notably, very few research articles have either reported, or discussed, the formation of amyloid fibrils by hyperthermophile-derived ultrastable proteins20-22. This is understandable, given the extreme structural stability of such proteins and their resistance to undergoing either unfolding or conformational changes. Thus, our interest in this paper is to examine amyloid fibril formation by such proteins, in particular, through the examination of the effect of the surfactant, SDS. SDS and amyloid fibril formation. Some years ago, a report demonstrated that the surfactant, SDS, at millimolar or sub-millimolar concentrations, is capable of inducing aggregation and amyloid formation in 25 diverse mesophilic proteins when the pH of the protein’s environment is lower than the isoelectric point (pI) of the protein, but not when the pH is either equal to, or above, the protein’s pI23. With the bulk of these proteins, it was shown that aggregation could be clearly observed when the pH of the buffer was two units of pH below the pI, or even lower. The report suggested that the effect of low concentrations of SDS involves a greater role for electrostatic interactions than hydrophobic interactions. In the present study, we tested five different hyperthermophile-derived ultrastable proteins from Pyrococcus furiosus in respect of their susceptibility to undergo fibril formation under entirely similar conditions. It may be noted that all five proteins have previously been studied and confirmed to be ‘ultrastable’ in nature, 5


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

being resistant to unfolding at room temperature by either 6 M Gdm.HCl or 8 M urea. Data regarding three of these proteins, namely aminopeptidase (PfuAP), rubredoxin (PfRd), and triosephosphate isomerase (PfuTIM) is already available in the published literature24-26. The fourth and fifth proteins, amylase (PfuAMY), and argininosuccinate lyase (PfuASL), too have been confirmed to be ultrastable (unpublished observations). In addition, we also studied a sixth protein, PEPM50, a metalloprotease encoded by the genome of the thermophile bacterium, Thermotoga maritima. Using spectroscopic and microscopic techniques, our focus here was on asking whether SDS is capable of inducing amyloid fibril formation in these proteins, and also on whether such an ability is dependent on the molecular weight, or quaternary structural status, or structural stability of the protein, and also on whether the CMC of SDS and the net surface charge on the protein are relevant, in these respects. The factors that could be clearly discerned to be important were (a) the number of residues on the protein’s surface which are capable of carrying positive charge, and (b) the number of residues actually carrying positive charge at a given pH.

Materials and methods Proteins were cloned, expressed and purified as described below. Sequence data and predicted physical-chemical data for all proteins is shown in the Supplementary Data file (Supplementary Table S1). Sodium dodecyl sulphate (SDS), Thioflavin T (ThT) and Congo Red (CR) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents used were of analytical grade. 6


Page 6 of 55

Page 7 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Cloning and purification of recombinant proteins. Genes encoding PfuAMY, PfuAP, PfuASL, PfRd and PfuTIM were already cloned and available in the lab, with information regarding some of these available in the literature24-26. PfuAMY and PfuASL were cloned between the NdeI and Xho I sites of the pET 23a vector and expressed in BL21starDE3plysS cells. The pET 23a vector encodes a 6xHis affinity tag at the 3’ end of the multiple cloning site, resulting in proteins bearing the poly-histidine affinity tag at their C-termini. PfRd, PfuAP and PfuTIM were cloned between the Bam HI and Hind III sites of the pQE30 vector (Qiagen) and expressed in M15[pREP4] cells. The pQE- 30 vector encodes a 6xHis affinity tag at the 5’ end of the multiple cloning site, resulting in these proteins bearing the poly-histidine affinity tag at their C-termini. The proteins were purified using standard immobilized metal affinity chromatography (IMAC) procedures (Qiagen), using Ni-NTA resin under native and non-denaturing conditions. The gene encoding PEPM50 (NCBI Reference Sequence- YP_008990348.1) was cloned from the genomic DNA of Thermotoga maritima through polymerase chain reaction (PCR) amplification and cloned between the NdeI and Xho I sites of the pET 23a vector and expressed in BL21starDE3plysS cells. Protein was extracted through heating of the lysate obtained after cell sonication at 80 0C, precipitating other proteins and leaving the supernatant enriched in PEPM50, which was then purified through standard IMAC procedures, with the exception being that 1 M NaCl was used during lysis and washing whereas normally 300 mM NaCl is used.

Protein concentration determination. Proteins stock solutions were prepared in 20 mM sodium phosphate buffer, pH 7.4. Protein concentrations were determined spectrophotometrically, using the following molar extinction coefficients at 280 nm on a Cary 50 Bio UV-Visible 7


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

spectrophotometer: 154715 M-1 cm-1 (PfuAMY), 42400 M-1 cm-1 (PfuAP), 27830 M-1 cm-1 (PfuASL), 14300 M-1 cm-1 (PfRd), 9770 M-1 cm-1 (PfuTIM) and 50420 M-1 cm-1 (PEPM50). Stock solutions of proteins were further diluted in buffers of desirable pH for experiments.

Two-dimensional gel electrophoresis and determination of pI. Two-dimensional gel electrophoresis was carried out by first loading 150 µg of protein on isoelectric focusing (IEF) strips of pI range from pI 3.0 to pI 10.0 (Ready Strip IPG strips, sourced from Bio-Rad) and performing isoelectric focusing on the Ettan IPGphor 3 system from GE. Thereafter, the strips were loaded onto SDS-PAGE (12% or 15% acrylamide) and electrophoresed along with molecular weight markers (Precision Plus Protein Dual Color standards from Bio-Rad) on apparatus sourced from GE, prior to staining and visualization.

pH measurements. pH measurements were carried out on a Metrohm-827 pH meter. Stock solutions of proteins were prepared in 20 mM sodium phosphate buffer, pH 7.4. Different salts, namely, sodium phosphate for pH 7.0 and 7.4, sodium acetate for (pH 4.5 and 4.0) and glycine HCl for (pH 3.5 and 3.0) were used for generating buffers of different pH values, and the molarity was maintained at a value of 20 mM in all cases. All buffers were filtered through a 0.22 µm MF-Millipore membrane filter before use.

Turbidity measurements. To assay SDS-induced aggregation, the turbidity of samples was measured in the absence and presence of varying concentrations of SDS at pH 7.4, 7.0, 4.5, 4.0, 3.5 and 3.0, by monitoring of changes in absorbance at 350 nm on a Cary 50 Bio UV-Visible spectrophotometer in a cuvette of 1 cm path length. Prior to turbidity measurements, all proteins 8


Page 8 of 55

Page 9 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

were pre-incubated with different concentrations of SDS for 12 h at pH 7.4, 4.5, 4.0, 3.5 and 3.0. The final concentration of protein was kept constant at a value of 5 µM, for all samples and experiments.

Anisotropy Measurements. Steady-state fluorescence anisotropy measurements were performed on a Fluoromax-4 (Horiba Jobin Yvon, NJ) spectrofluorimeter, for final protein concentrations of 5 µM. Samples were excited with light of 295 nm and emission was recorded at 345 nm, with excitation and emission slit widths of 2 and 10 nm, respectively. The steady-state fluorescence anisotropy (rss) measurement is given by the following relationship: rss=(I║-I┴G/(I║+2I┴G)

(1)

where I|| and I┴ are the fluorescence intensities collected, respectively, with the polarizer in the emission light path being in parallel, or perpendicular, geometry with respect to the polarizer in the excitation light path. The perpendicular components were corrected using a G-factor. The standard error was estimated from at least ten independent measurements. All samples were incubated overnight at room temperature, prior to measurements.

Thioflavin T (ThT) fluorescence measurements. ThT was dissolved in Milli-Q water and filtered through a 0.45 µm PVDF syringe filter (Millipore Milex-HV). The concentration of ThT was measured and determined by absorbance measurements based on a known molar extinction coefficient of 36000 M-1 cm-1 at 412 nm. Protein samples (5 µM) in the absence, or presence, of varying concentrations of SDS were incubated for a duration of 12 h, after which ThT (5 µM) was added to all samples. Prior to measurements, aggregated and non aggregated samples were incubated with ThT for 30 min in the dark, and the samples were agitated to resuspend any 9


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sedimented protein. ThT fluorescence spectra were recorded on

Page 10 of 55

a Fluoromax-4

spectrofluorimeter (Horiba Jobin Yvon, NJ) by exciting samples at 440 nm and monitoring emission in the range of 450–600 nm, using excitation and emission slit widths of 1 nm and 5 nm, respectively.

Assay of kinetics of increase of ThT fluorescence intensity. The kinetics of SDS-induced amyloid fibril formation was monitored though measurement of ThT fluorescence. Aggregation reactions were set up in various buffers of 20 mM concentration, with pH values selected to be two units below, and two units above, the pI of the respective proteins, either in the presence, or in the absence, of 1.0 mM SDS. ThT fluorescence development kinetics were monitored under three different conditions using 5 µM protein and 1.0 mM SDS. These conditions were: (1) protein and SDS being present in the same buffer of pH two units below the pI, (2) protein and SDS being present in the same buffer of pH two units above the pI (as an ‘SDS control’), and (3) protein (with no added SDS) in buffer of pH two units below the pI (as a ‘pH control’). In all these incubations, ThT was present at a concentration of 5 µM. ThT fluorescence intensity was measured as a function of time in a 1 cm path length (3 ml) cuvette on a Fluoromax-4 spectrofluorimeter (Horiba Jobin Yvon, NJ), with excitation and emission wavelength fixed at 440 and 485 nm, respectively, using excitation and emission slit widths of 1 nm and 5 nm, respectively.

Congo Red (CR) dye binding. CR was added in Milli-Q water and shaken until dissolved, and filtered through a PVDF (0.45 µm) syringe filter (Millipore Milex-HV). Actual concentrations of CR were measured through monitoring of absorbance at 498 nm, based on a molar extinction 10


Page 11 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

coefficient of 45000 M-1 cm-1 in Milli-Q water. All proteins (5 µM) were incubated in the absence and presence of 1.0 mM SDS for 12 h at a pH which was two units below the pI. The SDS-induced aggregates obtained were centrifuged at 5000 rpm for 5 minutes, following which the supernatant was discarded and the precipitate re-suspended in 20 mM phosphate buffer of pH 7.4. The re-suspended aggregates and non-aggregated samples were then incubated with CR (5 µM) for 30 min in the dark. Absorbance spectra of incubated protein samples were recorded between 400–700 nm on a Lambda 25 UV-Visible spectrophotometer (Perkin Elmer), in a cuvette with a path length of 1 cm. The control spectra of CR alone in the buffer and in the presence of 1.0 mM SDS were also collected.

Circular Dichroism Spectroscopy. CD measurements were carried out on a MOS-500 spectropolarimeter (BioLogic Science instruments, Claix, France). All measurements were performed at 25 0C with a speed of 100 nm min-1 and using a detector response time of 1 second. CD spectra were recorded in the wavelength range of 200-250 nm by averaging over five spectral collections. The protein concentration, i.e. 5 µM, was kept constant in all samples. The data was plotted as mean residual ellipticity (MRE) vs wavelength, using the formula : MRE= θobs (mdeg) x 100 x M.R.W/c(mg/ml) x l (cm)

(2)

where θobs is the ellipticity measured in millidegrees, M.R.W is the mean residue weight obtained by dividing the protein’s molecular weight by the number of amino acid residues, l is the path length of the cell in centimeters and c is the concentration of the protein in milligrams per milliliter.

11


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 55

Atomic Force Microscopy (AFM) Imaging. A Nanowizard II multimode atomic force microscope (JPK instruments) was used for imaging protein aggregates. All proteins (incubated overnight in the presence of 1.0 mM SDS in a buffer of pH two units below the proteins’ respective pIs) were taken in aliquots (15 µl) and placed on freshly mounted mica on glass slides. After 10 minutes the mica was washed multiple times with Milli-Q water to remove unbound protein, and dried overnight at room temperature. Imaging was performed with a digital nanowizard-3 head and controller. To avoid the effect of lateral forces, all measurements were carried out in the ‘tapping’ mode. We used hyperdrive cantilevers with force constants of ~250 kHz. Images (512 x 512 pixel scan) were collected at the scan rate of 1-2 lines/s, using a drive frequency of 285 kHz.

Results and Discussion Formation of aggregates upon exposure of ultrastable proteins to sub-millimolar SDS. Hyperthermophile-derived ultrastable proteins are generally very stable and do not ordinarily display aggregate- or amyloid-forming tendencies under any in-vitro or in-vivo conditions, with very few exceptions. Some 2-3 exceptions have already been mentioned in the introduction. Typically, extremely harsh conditions are required, e.g., with the Pyrococcus furiosus methionine aminopeptidase, fibril aggregate formation requires the presence of the denaturant, Gdm.HCl, at a concentration of 3.37 M, as well as a very low pH of 3.3120. No previous reports mention any surfactant-induced aggregate or amyloid fibril formation by ultrastable hyperthermophile-derived proteins. We explored the effects of sub-millimolar and millimolar SDS concentations on the conformational states and aggregation behavior of five Pyrococcus furiosus proteins and one Thermotoga maritima protein in buffers of pH fixed two units below, 12


Page 13 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

and/or two units above, the individual proteins’ pI values, at 25 °C. SDS is an amphipathic molecule containing a negatively charged head group and a 12 carbons-long hydrophobic tail which can interact with proteins by utilizing both its hydrophilic and hydrophobic moieties. We found that all six proteins almost immediately form amyloids in the presence of very low (0.1 4.0 mM) concentrations of SDS, when the pH is below the pI, but not when the pH is above the pI (under which conditions, no concentration of SDS could elicit aggregate-formation). The rapid nature of the aggregate formation is characterized in a subsequent section. The occurrence of SDS-induced aggregation when the pH is below (but not equal to, or above) the pI suggests that the critical causal factor could be the occurrence of electrostatic interactions between the positively-charged states of the protein molecules and the negatively-charged states of SDS molecules. The various ways in which aggregation and amyloid formation were studied, and confirmed, in this regard, are detailed below.

Development of turbidity. As seen in Figure 1A, the optical density profiles of solutions of all five proteins in the presence of SDS (0.1-10.0 mM) at 350 nm (O.D350) are clearly indicative of the development of turbidity owing to protein aggregation, over a smaller range of SDS concentrations varying from 0.1 to approximately 4.0 mM SDS, when the buffer is of pH two units below the protein’s pI. However, as seen in Figure 1B, when the pH of the buffer is two units of pH above the pI, rather than two units of pH below the pI, there is no substantive change in the O.D350 value at any concentration of SDS between 0.1 and 10.0 mM SDS, with respect to the basal O.D350 value measured for an SDS concentration of 0.0 mM. All five hyperthermophile-derived proteins possess high numbers of charged residues, as can be seen in Supplementary Table S1. When the pH of the protein’s environment is below the pI, it may be 13


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 55

surmised, the side chains of arginine, lysine and histidine residues, being protonated, would be likely to interact with the negatively charged head groups of SDS molecules, even while the hydrophobic tails of such SDS molecules could be expected to strongly repel water molecules from those regions of the protein to which the SDS molecules are bound. The lack of aggregation (and consequently, of turbidity) upon incubation of proteins with SDS in environments with pH two units above the pI suggests that SDS induces aggregation when the proteins are in cationic state, but not when they are in anionic state.

Changes in intrinsic fluorescence (tryptophan) anisotropy . Intrinsic fluorescence anisotropy is a method of choice for the study of the rotational motion(s) of tryptophan (Trp) residue(s) in proteins. Steady-state fluorescence anisotropy (rss) measurements of Trp fluorescence for any protein yield average time-integrated values indicative of the extent of rotational motion(s) occurring in Trp residue(s) in the excited state, with this being dependent on both fluorescence lifetime(s) and the rotational correlation time(s) of the residue(s)27. The rotations of Trp residues (in particular, those which are partially or wholly buried within proteins) tend to be strongly correlated with protein size and rotational motions of the protein as a whole, whereas motions of Trp residues on a protein’s surface are often not quite as strongly correlated, since the side chains of such residues are potentially free to rotate around the bonds that join them to their main chain alpha carbons. Therefore, protein aggregation is often accompanied by changes in rss values, since any already-restricted rotational motions of Trp residues tend to be further restricted by the association of protein chains, yielding larger and more slowly rotating entities. Figure 1C shows the anisotropy changes seen upon incubation of all five proteins with different SDS concentrations (0.1-10.0 mM), in buffers of pH two units below the pI. The anisotropy values of 14


Page 15 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

PfuASL, PfRd and PfuTIM are clearly seen to increase, peak and then decrease over the SDS concentration range of 0.1-4.0 mM SDS, but remain close to the baseline anisotropy value at 0.0 mM SDS at all higher concentrations in the range of 4.0-10.0 mM SDS. The increase in the rss values of these three ultrastable proteins at low SDS concentrations confirms that they have undergone aggregation, as already indicated by the turbidity data presented in a previous figure (Figure 1A). Interestingly, with the remaining two proteins, i.e. PfuAMY and PfuAP, the anisotropy was actually seen to decrease (rather than increase) with respect to the basal value observed at 0.0 mM SDS, over the same concentration range of 0.1 - 4.0 mM SDS in which rss values were observed to increase for the remaining three proteins. At present, we do not have a sufficiently clear explanation for this anomaly. However, we venture to speculate as follows. PfuAMY and PfuAP have far larger numbers of Trp residues in their sequences than the other three proteins (see Supplementary Table S1). It is conceivable, therefore, that the presence of the majority of these Trp residues on the surfaces of aggregates could (a) allow them to possess degrees of rotational freedom which are not available to Trp residues that are otherwise buried, and (b) therefore, dominate the averaged anisotropy which is measured for all tryptophans in such steady state measurements. Control experiments (data not shown) confirm that none of these effects are seen when the pH values of the buffer(s) used are above the proteins’ pI values, i.e., neither increases nor decreases are seen in Trp fluorescence anisotropy.

Binding and fluorescence of ThT. From the turbidity and fluorescence anistotropy data presented above, ultrastable proteins do appear to be caused to aggregate by very low concentrations of SDS in the sub-millimolar to millimolar range. We performed ThT binding-induced fluorescence experiments with these aggregates. ThT is a benzothiazole dye, which is perhaps the most 15


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 55

utilized of all dye probes employed for the detection of amyloid structure formation both in vivo and in vitro. ThT is believed to rapidly bind with amyloid (cross-beta) microstructures and form highly fluorescent complexes28; notably, although it can bind to exposed β-sheet edges, and extended β-sheet surfaces, it is thought to mainly bind to laminated “steric zipper” interfaces between β-sheets and to cross-β sheet architectures29. ThT binding and fluorescence is thus held to be an important indicator of the degree of amyloid-like structure present inside an aggregate, although ThT-binding species can also manifest initially in an apparently-amorphous morphology with fibrils being observed only at later points in time, when smaller aggregates associate to form long discernible amyloid fibers. Figure 1D shows the intensities of fluorescence of ThT (5 µM) upon addition to aggregated samples of all five ultrastable proteins, produced (as earlier described) using SDS and buffers of pH two units below the proteins’ pI values. In all cases, it is evident that ThT fluorescence is significantly high for concentrations of SDS between 0.1 mM and 4.0 mM. Control data (not shown here) confirmed that no effect was seen with buffers of pH above the pI values of these protein(s), when ThT was added to the proteins, demonstrating that ThT does not bind at all to the soluble forms of any of these proteins. The data confirms that SDS causes all five ultrastable proteins to associate into aggregates with amyloid-like features, and the capability of binding to ThT and causing ThT to fluoresce over the exact same range of SDS values in which turbidity and increase in anisotropy are observed. Supplementary Figures S1A-S1E show the actual ThT fluorescence spectra for each protein in the presence of 0.0, 1.0, 1.5, 2.0, 3.0 and 5.0 mM SDS, along with spectra collected using 1.0 mM SDS for several ‘controls’ which lacked either protein, or SDS, or included SDS and/or ThT at the same pH values, i.e., two units of pH below the proteins’ pI values. From the data, it is clearly evident that there is a dose-dependence of the effect of SDS on 16


Page 17 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ThT binding and fluorescence over the entire wavelength range of the ThT spectrum. This indicates that aggregation and amyloid formation occur only over the range of 0.1 mM to 4.0 mM SDS, and when the pH is below the pI, and neither at higher SDS concentrations, nor at pH values which are above the pI. The question that now arises concerns how fast the process of aggregate- and/or amyloid-formation occurs, with these proteins. This is examined and described in the next section.

Rapid kinetics of SDS-induced aggregation monitored through ThT fluorescence. The kinetics of formation of amyloid-like (ThT-binding) features in the SDS-induced aggregates was observed by monitoring the rise in ThT fluorescence in the presence of 1.0 mM SDS at 485 nm with the passage of time (between 0 and 600 seconds, for four of the five proteins, or between 0 and 12000 seconds for PfuTIM), prior to saturation of the signal, using buffers of pH two units below the pI for each of the five proteins. The kinetics data is shown in Figures 2A-2E, in panels corresponding to the equilibrium data shown in Supplementary Figures S1A-S1E. In these figures, in all cases, the ThT fluorescence is seen to have begun to rise very rapidly, within the dead-time of the ThT mixing. For comparison, data is also shown for samples exposed to 1.0 mM SDS at a pH (two units) above the pI, and with no SDS present at a pH two units below the pI, to establish the rapid rise in ThT fluorescence in the experiments involving SDS-induced aggregates relative to the baseline(s), and to demonstrate once again that the effect is only seen when the proteins carry a net positive charge, i.e,. when they are in cationic state. It is interesting that there is no apparent ‘lag’ phase observed in the rise in ThT fluorescence, indicating the possible occurrence of downhill, non-nucleation-dependent polymerization, or ‘isodesmic’ polymerization. As soon as SDS is added, i.e., at the zero (seconds) timepoint, there is already 17


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 55

considerable ThT fluorescence seen in most of the samples, probably owing to the moderately low time-resolution of the data collection. The fluorescence rises gradually to saturate over a few hundreds of seconds which, for all intents and purposes, is tantamount to being ‘immediate’ or ‘instantaneous’; indeed, startlingly so in respect of such ‘ultrastable’ proteins for which such rapid kinetics of aggregation would be quite unanticipated. Notably, four of the five proteins, i.e., PfuAP, PfuAmy, PfuASL and PfRd were observed to bind to ThT extremely rapidly and reach saturation within 2-3 minutes of the addition of SDS, whereas with PfuTIM the process was slower and took nearly 2 hours to reach saturation. From the kinetics it can be concluded that protein monomers probably directly assemble into aggregates with amyloid-like (i.e., ThTbinding) features, although the need still remains to establish whether clearly discernible amyloid fibrils already form by this time point. Notwithstanding this small caveat, the data appears to be similar to that reported earlier for a mesophile-derived protein, beta-2-microglobulin (β2m), for which very fast kinetics of amyloid fibril formation in the presence of 0.25 mM MPPA or 0.5 mM SDS has previously been described by other authors,

where the increase in ThT

fluorescence intensity was similarly observed to show no lag phase, with saturation occurring only after the passage of 24-36 hours30. In contrast, as already mentioned, saturation in the case of PfuTIM occurred within 2 hours and for the remaining hyperthermophile proteins it occurred in a matter of minutes. Perhaps the lag phase in our studies (if any) was very small, and below the limit of detection and time-resolution. Several other molecules are known to promote amyloid formation, apparently without the formation of a nucleus. Heparin, for example, modulates amyloid formation in apomyoglobin without the formation of a nucleus and no lag phase can be detected31. Similarly, heparin also stimulates muscle acylphosphatase to oligomerize without the formation of a nucleus32. A small protein, barstar, forms amyloids 18


Page 19 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

apparently without the formation of a nucleus, with the ThT-binding kinetics similarly showing no recognizable lag phase and with data being fitted to a single exponential rise33,34. In all of these cases, amyloid formation has been called ‘isodesmic’, i.e., occurring without the necessary formation of any intermediate species and in a non-nucleation-dependent manner, through the direct assembly of monomers possessing altered aggregation-prone conformations. In our case, we would similarly like to call the SDS-induced aggregation and polymerization ‘isodesmic’; however, we shall not venture to call the amyoid formation itself isodesmic, as we have no evidence to establish that this is so.

Binding and spectral shift in Congo Red (CR). We confirmed further that the aggregates giving rise to turbidity and rise in fluorescence anisotropy are amyloid-like in nature, by using another dye, Congo Red (CR). CR molecules are largely specific in binding cross-β-sheet structures in amyloid fibrils and they bind along the fibril axis. Binding is initially electrostatic, because of interactions between the negatively charged sulphate groups of CR and the positively charged amino acid residues of proteins35. Subsequently, CR is thought to intercalate between the antiparallel β-pleated sheets of amyloid fibrils through aromatic-aromatic, pi-pi ring-stacking interactions. CR binding to β-sheets in amyloid fibrils is known to result in a red-shift in CR’s absorbance spectrum. We performed CR binding studies on the five proteins induced to aggregate in the presence and absence of 1.0 mM SDS at different pH values, by producing the aggregates and then re-suspending them in buffer of physiological pH, prior to exposure to CR. The results are shown in Figures 3A-3E. The absorption spectrum of CR alone is shown in Figure 3F, both in the absence and presence of SDS, to rule out any possible effect of residual protein-bound SDS on the spectral characteristics of CR. The control data in Figure 3F is also 19


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 55

included for comparison, to establish whether a red-shift in CR absorption is otherwise observed with the aggregates re-suspended in buffer of physiological pH. It may be noted that the resuspension of aggregates was necessitated by the fact that CR is pH-sensitive and cannot be used below pH 5.0 in such experiments, because the absorption spectrum of CR changes without any external influences at acidic pH. The control CR spectrum in Figure 3F shows two bands; one at ~ 350 nm, owing to a π-π* transition of an -NH group and another at ~ 480 nm owing to a π-π* transition of an azo group. We monitored the ~480 nm absorption band. The absorbance spectra of CR in the presence of the five proteins, collected in the absence of SDS at physiological pH, yielded maximum absorbance values at ~490 nm. In contrast, in the presence of 1.0 mM of SDS and aggregates of the five protein(s), at physiological pH, the absorbance maxima of CR were seen to be prominently red-shifted in all the hyperthermophile-derived protein aggregates. The shifts in the absorbance maxima of CR suggest that the SDS induced aggregates of the five hyperthermophile proteins have CR binding-capable β-based microstructures. From the dye binding assays involving ThT and CR, we venture to propose that the SDS-induced aggregates of the hyperthermophile proteins have amyloid-like features, at least at the level of microstructure involving the reorganization of the polypeptide backbone(s). It may be noted that a similar shift of absorbance maximum of CR has been reported for the aggregation of Aβ (25-35)36.

Confirmation of amyloid morphology by atomic force microscopy. The morphologies of SDSinduced aggregates formed by the five ultrastable proteins were studied by atomic force microscopy (AFM). AFM is a technique frequently used to distinguish between amyloid-like and other (e.g., amorphous) morphologies of protein aggregates. The shapes of amyloids can be worm-like, rod-like, ribbon-like, bead-like or ring-like and also long and straight fibrils37-39. 20


Page 21 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

AFM images of the aggregates of two representative ultrastable proteins, PfuAP and PfuASL, are shown in Figures 4A and 4B, following exposure to 1 mM SDS at pH two units below pI. Both proteins can be seen to form amyloid fibrils along with some amorphous deposits, and the amyloid morphology is the same with both proteins (long straight fibrils). The average heights of both PfuAP and PfuASL fibrils are ~ 2.5 ± 0.1 nm. While the long and straight fibril morphology was also seen with a mesophile protein β2M when proteins were incubated at extremely acidic pH (below 2.0) in the absence of salts, in that case the aggregate-formation was nucleationdependent, involving a lag phase40. It is generally assumed that the long–straight fibril is formed in a nucleation-dependent manner, and takes time to form. Therefore, it must be mentioned here that the AFM images shown in Figures 4A and 4B were collected for samples incubated overnight with SDS. With most proteins incubated at extremely acidic pH with denaturants, amyloid formation takes days if not weeks to occur. Thus, it appears that fibril formation occurs in a more facile manner with these hyperthermophile-derived proteins. To address the question of whether amyloid formation can be seen at even shorter time points, data is shown in Figures 4C and 4D, for SDS-induced aggregates of PfuASL examined after a mere 30 minutes of incubation with 1.0 mM SDS. Following deposition and drying, PfuASL incubated for 30 minutes with SDS was found to have formed long and straight structures along with some amorphous structures which were, of course, not as mature as those shown in Figure 4A and 4B. Notably, at an even shorter time point of 10 minutes, i.e., with deposition of aggregated protein on mica substrates after just 10 minutes of incubation with SDS, we were unable to see any long fibrillar structures. We could see only bead-shaped aggregates (Supplementary Fig S2). The observation of long and fibrillar amyloid-like structures

at later time-points (i.e., after 30

minutes) for an experimental system in which ThT-binding is observed to saturate within 2-3 21


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 55

minutes can potentially be explained as follows. The initial structures that are rapidly formed, which bind to CR and ThT (as well as display ThT fluorescence) but which display non-fibrillar, bead-like morphology on AFM probably contain cross-beta structures formed by (and between) polypeptide chains but these have not yet assembled into discernible amyloid fibrils. The fact that amyloid fibrils are visible at subsequent time-points of deposition indicates that amyloid-like polypeptide chain arrangements could be already present in the initially-formed aggregates which, however, have not yet associated further to grow into visible fibrils. The important point to note, however, is that ThT-binding saturates long before the long fibrillar morphology can be seen, indicating that the bead-shaped aggregates too are amyloid-like in nature.

A far-UV CD minimum at ~230 nm is associated with these amyloids. Circular Dichroism (CD) is a useful technique to determine secondary structural content, and to monitor secondary structural changes in proteins. We monitored changes in the secondary structures of all five ultrastable proteins in the absence, and presence, of 0.0, 1.0, 1.5, 2.0, 3.0 and 5.0 mM SDS, in buffers of pH two units below and two units above the proteins’ pIs, as shown in Figure 5A-5E. Control far-UV CD spectra of four of these proteins (PfuAMY, PfuAP, PfuASL and PfuTIM) in the native state, i.e., in the absence of SDS, exhibited two minima at 208 nm and 222 nm, characteristic of the predominance of α-helical structure. These two minima dominate the CD spectrum of any protein that contains a significant amount of helical content, because the mean residue ellipticity (MRE) signal measured in CD spectroscopy per residue in alpha helical structures is 5-10 times stronger than the CD signal measured per residue in beta sheet structure. The control CD spectrum of the fifth protein, PfRd, is more unusual, and characteristically shows two minima, one at ~202-205 nm and the other at ~225 nm25; these minima owe to mixed 22


Page 23 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

contributions from beta sheet, random coil and polyproline type II (PPII) structure CD signals, as well as to excited state induced-CD transitions of PfRd’s six aromatic residues (W3, Y10, Y12, F29, W36, F48) which form an aromatic cluster within PfRd’s hydrophobic core. In control experiments, the MRE values of PfRd’s two bands were of reduced intensity at pH two units below the pI, i.e., at pH 3.0; however, with the other proteins, the MRE values were identical at pH two units below pI and two units above pI, consistent with their ultrastable character which brooks no evident structural change with change of pH over a broad range. Upon addition of low concentrations of SDS, the secondary structures of all five proteins can be seen to have been transformed into a spectrum with a ~230 nm minimum, indicative of a β-based microstructure formed in the presence of 1.0, 1.5 and 2.0 mM of SDS, when the pH was two units below the pI. The minima at 208 and 222 nm of the four proteins dominated by alpha helical structure were all transformed into a single observable minimum at ~230 nm in the presence of sub-millimolar and millimolar concentrations of SDS, but the exact position of the ~230 nm minimum was different for each of the four proteins, as was the shape of the altered CD spectrum. A similar transition to a ~230 nm minimum was noted in the case of PfRd as well, in the presence of 1.0, 1.5 and 2.0 mM of SDS, and the position and shape of the ~230 nm minimum was somewhat different from that applying to the other four hyperthermophile proteins. In the case of PfRd, a single peak was centered closer to ~235 nm. Notably, at higher SDS concentrations, particularly ~5.0 mM SDS and above, the CD spectra of all five hyperthermophile proteins turned into spectra dominated by an overt α-helical CD signature. It is known that, at higher concentrations, SDS induces helical structure in several different proteins41,42. Ostensibly, this owes to the binding of SDS, and a possible mechanism of helixinduction involving intra chain hydrogen bonding. 23


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 55

Differences between the behavior of ultrastable and ordinary proteins. The really surprising conclusion of the above studies was the finding that despite the well-known, inordinately-high structurally stabilities of hyperthermophile-derived proteins, in general, and the demonstrated ‘ultrastability’ of the five proteins studied here, in particular, none of these five proteins displayed any remarkable differences in behavior, in comparison with other mesophile-derived proteins for which SDS-induced structural changes and aggregation has earlier been reported for low concentrations of SDS at pH values below the pI. At most, perhaps, what can be said is that the range of SDS concentrations over which the amyloid-inducing effect can be seen with these hyperthermophile proteins (0.1-4.0 mM SDS) is considerably broader than that which has been reported for mesophile albumins (0.1-2.5 mM SDS)43. Beyond this, in terms of data which can be compared across the mesophile and hyperthermophile domains, there is very little in terms of notable differences. Amongst the hyperthermophile proteins, however, some formed amyloids at lower SDS concentrations than others (and involving a smaller range of SDS concentrations), the surprise being that PfRd showed the greatest vulnerability to SDS-induced amyloid formation, despite being one of the most stable proteins known to man. Below, we discuss how an analysis of these ‘unremarkable’ differences points towards further evidence of the amyloid-inducing effect of SDS being largely electrostatic.

A control experiment with a thermophile protein with an alkaline pI. We carried out an additional experiment involving a protein from a thermophile bacterium, Thermotoga maritima; a putative metalloprotease known as PEPM50 which has been produced and studied by us and found to undergo structural melting above 78 ºC (data not shown). Our reason for performing 24


Page 25 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

this experiment was that PEPM50 has a pI in the alkaline range, close to ~9.5, whereas the pI values of all five hyperthermophile-derived ultrastable proteins is in the acidic range, between 5.0 and 6.1. To exclude specific effects at a pH of 3.0-3.5 or 4.0, it was necessary to also examine whether the effect is seen at higher pH values. After confirming the alkaline pI of PEPM50 through isoelectric focusing, we subjected it to similar treatment with SDS, mainly to examine whether the same SDS-induced aggregation effects are seen even at higher pH. Data similar to those presented earlier in this paper (i.e., turbidity, ThT-binding and CD spectra) are shown for T. maritima PEPM50 in Figure 6, at pH values of 7.0, 5.0 and 4.0, clearly establishing that even with this protein SDS has the same charge-dependent effects.

Verification of the isoelectric points (pIs) of the proteins studied. Since the discussion of the effects reported in this paper focuses critically on the interaction of negatively-charged SDS molecules with the positive charges on proteins present in solutions of pH above or below their pI, it is necessary to establish whether or not the actual pI values of the proteins are reasonably close to their predicted pI values. This becomes necessary because residues that are capable of carrying charge can sometimes exist in environments that are not conducive to the carrying of charge, upon chain folding and multimerization. We performed two-dimensional gel electrophoresis to determine the isoelectric points of all six proteins. The results are shown in Figure 7. Figure 7A shows the behaviour of PfuAMY and PfRd. PfuAMY is seen to be migrating close to the ~ 75 kDa molecular weight marker, suggesting that it migrates as a monomer. Interestingly, PfuAMY is also seen to have a very broad and dispersed isoelectric point profile, which is centred around a pI of ~ 5.3, close to the predicted pI of 5.4; it must be noted, however, that PfuAMY shows two distinct sub-populations within this broad profile, which are centred 25


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 55

around pI values of 4.1 and 6.4, respectively. In the same gel, PfRd is seen to migrate with an apparent molecular weight of ~ 12 kDa. PfRd has a much tighter distribution of charged species, centred around a pI of 4.7 which is higher than the predicted pI of 5.1. Figure 7B shows PfuASL migrating with an apparent molecular weight of ~ 50 kDa (monomer) with a minor subpopulation at ~ 100 kDa, owing presumably to residual dimeric species which are incompletely dissociated by SDS. PfuASL displays a tight distribution of charged species, centred at a pI of ~ 5.9, again somewhat higher than the predicted pI of 5.5. Figure 7C shows PfuAP displaying several populations, with the main population showing an apparent molecular weight of ~ 37 kDa (monomer) and an apparent pI of 7.2, considerably higher than the predicted pI of 6.1. The native state of PfuAP is a dodecamer of ~ 480 kDa, and some dodecameric PfuAP is seen at the top of the gel with a subpopulation displaying a pI closer to the predicted pI. Figure 7D shows PfuTIM with an apparent pI of 6.3, higher than its predicted pI of 5.7. PfuTIM also shows an apparent molecular weight of ~80 kDa, migrating as a tetramer with a degree of anomalous mobility. It may be noted that many proteins from hyperthermophile archaea and bacteria display anomalous mobility on SDS-PAGE if they are not boiled in the presence of SDS during preparation of samples for electrophoresis. Depending on the stability of the protein concerned, and the duration and temperature of exposure to SDS prior to electrophoresis, different fractions of population are observed to display either the correct mobility, or some anomalous mobility, owing to (i) incomplete dissociation of subunits, (ii) incomplete unfolding of dissociated subunits, or (ii) insufficient SDS-binding by incompletely unfolded chains. These various possibilities are associated with various gel-migration outcomes, because incomplete dissociation of subunits leads to retarded mobility, whereas incomplete unfolding of subunits leads to 26


Page 27 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

increased mobility (due to reduced hydrodynamic volume) and incomplete SDS-binding leads to retarded mobility. Since samples subjected to isoelectric focussing in the first dimension and SDS-PAGE in the second dimension are never boiled with SDS, but only exposed to urea which fails to unfold many hyperthermophile proteins, anomalous mobility is sometimes observed with such proteins. In the present instance, anomalous mobility is also observed with PEPM50 which remains mostly within the IEF strip, barely entering the gel. Within the IEF strip seen in Figure 7E, PEPM50 is observed to have a broad and dispersed charge distribution centred around a pI of 8.2. Supplementary Figure S3 shows another gel in which PEPM50 is observed to be similarly retained in the IEF strip, with a somewhat tighter dispersed charge distribution centred around a pI of 8.7. The predicted pI of PEPM50 is 9.5. Thus, the isoelectric focusing experiments reveal that PEPM50 has a pI which is almost one unit of pH below the predicted pI, whereas PfuAP has a pI that is almost one unit of pH above the predicted pI. In all other cases, the predicted and actual pI values are reasonably close to each other. Further, some proteins have tighter charge distribution profiles than other proteins, and some proteins display anomalous mobility owing to the lack of boiling with SDS while other proteins display the expected mobility.

The pre-CMC and (likely) post-CMC effects of SDS. In this study, it would appear that the effects of SDS can be seen even above the CMC of SDS (i.e., post-CMC), and not just at preCMC concentrations. Our contentions regarding this are based on reported conductivity measurements showing that the CMC of SDS in 20 mM glycine-HCl buffers of pH 2.0 at room temperature is ~ 1.2 mM

44

. We used 20 mM glycine-HCl buffers of pH values, 3.6, and 3.5, 27


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 55

respectively, for work with PfuAMY, and PfuASL. For these pH values, the CMC of SDS is anticipated to be about 2.0 mM, or maybe somewhat higher. Notably, though, with both PfuAMY and PfuASL, we were able to see the amyloid-inducing effect of SDS up to a concentration of 4.0 mM SDS, which would appear to be above the CMC, using turbidity, fluorescence anisotropy, and ThT binding and fluorescence data. Thus, for at least two of the five proteins, induction of amyloids was observed at SDS concentrations above the anticipated CMC of SDS. The question is whether this is significant and, if it is significant, exactly what it physically means in respect of SDS-induced protein aggregation. It may be noted that the CMC values of SDS have been repeatedly reported to be in the range of 1.0 mM SDS, and sometimes below or somewhat above this value. The CMC has been determined to be 1.0 mM in 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl at 37 °C. In several other studies, it has been reported that the CMC of SDS can be decreased by the presence of proteins. One experimental determination of the CMC of SDS based on the use of the fluorescence probe, pyrene, in the presence and absence of the protein alpha synuclein (αSN, at 20 µM protein concentration) at physiological pH, found the CMC to be 0.75 mM in the absence of αSN and lowered greatly (to 0.15 mM) in the presence of αSN. In other reports, the CMC of SDS was found to be around 0.1 mM in the presence of αSN in phosphate buffered saline (PBS) at 25 °C45,46. This indicates that, in all likelihood, most of our experiments have been conducted at SDS concentrations above the CMC. In contrast to this view, several authors have reported that they see the aggregation-inducing effects of low concentrations of SDS only below the CMC of SDS12,47,48. Below, we present our interpretation of this apparent conundrum which, however, is easily explained if one takes the CMC to be influencing the SDS-induced aggregation behavior but not absolutely determining it. 28


Page 29 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Firstly, we discuss the likely reason(s) for an apparent correlation of SDS-induced aggregation behavior with CMC, in the light of what we have learned in this paper, in terms of electrostatic interactions between SDS and proteins. The formation of micelles by SDS concentrates the negatively charged head groups of SDS into spheres of definite volume and surface area. It is likely that this reduces, for entirely geometric and steric reasons, the scope for the interaction of SDS with individual positively charged residues on proteins, because the micelles formed above the CMC (albeit negatively charged) cannot interact with proteins in a sufficiently-facile manner, owing to micellar curvature and other factors, as suggested in the schematic in Figure 8. In any micelle that manages to interact with a protein, for entirely geometric reasons, a large number of SDS molecules located on the micelle would become unable to interact with the protein associating with the micelle (because these molecules would be on the other side of the micelle, and no longer freely-diffusing monomers), whereas at preCMC concentrations all of these SDS molecules would exist in the form of free monomers, with each potentially remaining capable of participating in the binding to the protein. Ordinarily, therefore, at concentrations immediately flanking the CMC, the formation of micelles could be expected to reduce the available monomers of SDS which are free to bind to the protein, by titrating them away into micelles, with all subsequently added surfactant participating in the formation of micelles. It is possible that, with mesophile proteins which are not ordinarily endowed with large numbers of surface charges, these factors facilitate SDS-induced aggregation behavior at pre-CMC concentrations, and more or less prevent SDS from showing the amyloidinducing effect at post-CMC concentrations.

29


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 55

Next, in discussing what has been observed in the experiments presented here, it is possible that if the number of positively charged residues available on the protein’s surface - for interacting with any negatively charged SDS molecule - is substantially increased in comparison with other proteins, this could serve to somehow contort micelles, or ‘bleed’ SDS molecules away from micelles, or otherwise manage to bind more effectively and efficiently to

free monomers

remaining in solution under post-CMC conditions (depending on the nature of the equilibrium of dissociation and reassociation of SDS into micelles) in a more facile manner. This would be simply owing to the presence of charges at higher density on the protein’s surface, for hyperthermophile

proteins.

As

already

shown

in

Supplementary

Table

S1,

these

hyperthermophile proteins have very high numbers of surface charges.. Thus, it is likely that we have been able to see these effects even considerably above the CMC of SDS primarily because of the presence of very large numbers of Lys, Arg and His residues in the five hyperthermophile proteins, allowing far higher numbers of positive charges to be present in these ultrastable proteins (than is possible with mesophiles), in buffers of pH two units below the pI. To take this argument somewhat further, the understandably-low probability of a protein interacting with micellar SDS could probably be offset, and the total number of effective interactions be increased, by increasing the number and density of positive charges on a protein’s surface. Indeed, this is very likely to be the effect that we are seeing here, suggesting that the presence of enough positive charges on a protein molecule can locally offset the presumed unavailability of free SDS resulting from formation of micelles, at SDS concentrations immediately preceding the CMC (pre-CMC) and immediately succeeding the CMC (post-CMC). A predictable consequence of this argument, which is borne out by our data, is that one must observe a slight ‘postponement’ of the upper limit of the concentration range over which the 30


Page 31 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

effect of SDS is observed, extending it up to ~4.0 mM SDS as reported here, for hyperthermophile proteins possessing high numbers and densities of positive charges. However, we also emphasize that since we haven’t actually experimentally measured the values of CMC for different conditions, these arguments about the likelihood of our having observed SDSinduced aggregation above the CMC, based on CMC measurements (and CMC-lowering effects of proteins) made by other groups, must be taken to be merely conjecture, and not certainty, at this point.

Differences observed amongst the five ultrastable proteins supporting the possibility of post-CMC amyloid formation. All five ultrastable hyperthermophile proteins tested in this study have different intrinsic features, in terms of their secondary, tertiary and quaternary structures and functions (see Supplementary Table S1), although the five proteins do have in common the fact that their amino acid sequences evolved within the same (Pyrococcus furiosus) genome. As already shown in Figures 1-3 (but not yet discussed), the five proteins did show some significant differences in responses to SDS, although all were consonant with the interpretation outlined above. Below, we show how the differences in response amongst the five proteins also support our interpretations regarding what probably happens above and below the CMC. An analysis of the different proteins reveals that the observed effect is dependent on the numbers of positive charges present and available for interaction with SDS in each protein. The net charges of all five hyperthermophilic proteins were estimated at pH two units below the pI by using PROTEIN CALCULATOR v3.3 software (Table 1). At one end of the behavior spectrum, PfRd retains a net charge of around +12 at pH 3.1, and displays amyloid formation in the concentration range of 0.1 to 2.0 mM SDS, above which aggregate/amyloid formation is 31


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 55

clearly inhibited. At the other end of the behavior spectrum, PfuAMY forms amyloids in the concentration range of 0.1-4.0 mM SDS, ostensibly because the net charge on PfuAMY is more than seven times higher (around +86), in a buffer with pH two units below the pI. In the middle of the behavior spectrum, the other three proteins, PfuAP, PfuTIM, and PfuASL, form amyloids in the concentration ranges of 0.1-2.5, 0.1-2.5. and 0.1-3.0 mM SDS, respectively, which is broadly in accordance with the increasing numbers of positive charges present on these proteins in buffers of pH two units below the pI, for which the values are +46, +59 and +59 charges, respectively. From these observations, and interpretations, it can be proposed that SDS-induced amyloid formation is not dependent on protein stability, or structure, but rather on the net positive charge that can be present on each protein, at any value of pH, because the lower concentration of SDS inducing amyloids is the same for all hyperthermophile proteins whereas the ranges of concentrations over which these effects are seen do tend to vary considerably, and in accordance with the numbers of charges present, i.e., the greater the numbers of charges, the more likely the effect continues to be observed at higher SDS concentrations (approaching or exceeding the CMC). The predicted profile of titration of charge as a function of pH for all five hyperthermophile-derived P. furiosus proteins, and for T. maritima PEPM50,

is shown in

Supplementary Figure S4. Earlier, it has been reported that some mesophile-derived proteins (lysozyme and stem bromelain) form amyloids in the SDS concentration range of 0.5-1.0 mM SDS, and that fibril formation is suppressed at higher concentrations44. In the case of lysozyme, in the pH range of 4.0-1.0, amyloid formation was found to be induced in the broader concentration range of 0.1-2.5 mM SDS, presumably owing to lysozyme having a higher net positive charge at pH 7.0, in comparison to what obtains at pH 7.049. 32


Page 33 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

These interpretations lends further support to the argument that the aggregation- and amyloid-formation-inducing effects of SDS on proteins tend to be predominantly determined, at least initially, by electrostatic interactions between SDS and basic residues on the protein’s surface, with hydrophobic interactions only playing a subsequent (and subjugate) role, even as earlier suggested21, with this applying to all proteins, regardless of structural stability. Below the pI, basic amino acids (Arg, Lys and His) become protonated while negatively charged residues remain uncharged, and the protein has a net positive charge which interacts with the sulphate group of SDS; however, above the pI, each protein becomes negatively charged and repels the advances of SDS. Once the SDS-protein interaction has occurred, however, the hydrophobic tail of SDS must adversely affect protein-solvent interactions, causing enhanced conformational changes accompanied by greater protein-protein interactions, and this is probably what results in the formation of amyloid fibrils. Also, an SDS-bound monomeric protein would not be able to directly interact with (or form cross beta sheet structures with) other protein monomers in an ‘isodesmic’ fashion if the SDS were not replaced by protein-protein interactions. A schematic of this occurrence, in the form of a hypothesis, or model, is shown in Figure 9, in which an initial conformational conversion in monomeric proteins, upon SDS binding, is shown to transform into formation of amyloids which continue to remain bound to SDS (but only on their peripheries) with bound SDS serving in the interim to mediate interactions of a hydrophobic nature between SDS bound (and conformationally transformed) protein molecules. In summary, we can conclude that despite their stability, all hyperthermophile proteins tested here behaved like mesophile proteins with respect to their response to SDS. Our results provide some new mechanistic evidence for the interaction of proteins with SDS. SDS 33


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 55

electrostatically interacts with any protein in a cationic state. The CMC of SDS, while important, is not very important, except in that it prevents access of proteins to free SDS monomers and reduces the monomers available for binding. The net positive charge of proteins determines the concentration range of SDS in which the aggregation- and amyloid-inducing effects are seen. Highly charged proteins can tolerate higher concentrations of SDS before forming amyloid fibrils, presumably by locally bleeding SDS monomers from micelles, or contorting micelles. The SDS-induced amyloid fibril formation is independent of the molecular weight of the proteins, and their quaternary structures and structural stabilities, perhaps because the undoing of electrostatic interactions on a hyperthermophile-derived protein’s surface by SDS weakens these proteins sufficiently to make them as vulnerable to SDS as other proteins. In closing, we must mention that although our interpretation of the results relies almost entirely on the hypothesis that SDS elicits its effects based on the net positive charge (determined by the difference between pH and pI), it is also possible that some role is played by the protonation of acidic residues (especially in the critical region of pH between 3.0 and 5.0, supporting the ionization of residues Asp, and Glu). Virtually all the data (including that presented for PEPM50) shows that the major change in turbidity occurs in the pH range of 3.0 to 5.0. Therefore, it could very well be that the net effect derives from a combination of both ionization of acidic residues and the presence of a net positive charge, with the former being even more important than the latter. We have attempted to examine this further by determining how overall turbidity varies as a function of pH (by determining the total areas under the peaks in the turbidity plots in Figure 1) and as a function of predicted net positive charge. The results of this analyses are presented in Supplementary Figure 5 which, unfortunately reveals no trends that would settle this particular issue. 34


Page 35 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Acknowledgements The facilities provided by Indian Institute of Science education and Research (IISER) Mohali are gratefully acknowledged. J.M.K acknowledges IISER Mohali for financial support in the form of postdoctoral fellowship. P.S, K.A, N.K and P.K acknowledge doctoral fellowships from CSIR-India, UGC-India, UGC-India and IISER Mohali, respectively. The research groups of Dr. Bishwajit Kundu, Dr. Sabyasachi Rakshit, Dr. Goutam Sheet, and Dr. Samrat Mukhopadhay are acknowledged for help with AFM experiments, at different times. Dr. Preeti Bhora is thanked for help with two-dimensional gel electrophoresis. The referees (unknown) are thanked for insightful comments that helped to improve the paper.

Research Funding The

work carried out for this paper was supported by research grant MHRD-14-0064,

supporting the Centre of Excellence (CoE) for Protein Science, Design and Engineering (CPSDE) in Frontier Areas of Science and Technology (FAST), provided by the Ministry of Human Resource Development (MHRD), Govt. of India.

Supplementary information available. Supplementary Table S1: Physical, chemical and structural characteristics of the five P. furiosus proteins and T. maritima PEPM50. Supplementary Figure S1: Fluorescence emission spectra of ThT bound to the five P. furiosus proteins aggregates using different SDS concentrations. Supplementary Figure S2: Bead-shaped aggregates produced after 10 minutes of incubation of 35


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 55

PfuASL with 1.0 mM SDS. Supplementary Figure S3: Two-dimensional gel containing all six proteins used in this study within a single gel, reiterating the observation that PEPM50 remains largely trapped in the IEF strip at alkaline pI.. Supplementary Figure S4: Predicted charge vs pH titration profiles of all five P. furiosus proteins and T. maritima PEPM50. Supplementary Figure S5: Plots of SDS-induced turbidity as functions of pH and overall positive charge.

References: 1.

Szilagyi, A., and Zavodszky, P. (2000)

Structural differences between mesophilic,

moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey, Structure 8, 493-504. 2.

Rees, D.C. (2001) Crystallographic analyses of hyperthermophilic proteins, Methods. Enzymol. 334, 423-437.

3.

Matsui, I., and Harata, K. (2007) Implication for buried polar contacts and ion pairs in hyperthermostable enzymes, FEBS. J. 274, 4012-4022 .

4.

Watanabe, K., Hata, Y., Kizaki, H., Katsube, Y., Suzuki, Y. (1997) The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization, J. Mol. Biol. 269, 142-153.

5.

Kumar, S., Tsai, C. J., and Nussinov, R. (2001)Thermodynamic differences among homologous thermophilic and mesophilic proteins, Biochemistry 40, 14152-14165.

6.

Ji, S. R., Wu, Y., and Sui, S. F. (2002) Cholesterol is an important factor affecting the membrane insertion of beta-amyloid peptide (A beta 1-40), which may potentially inhibit the fibril formation, J. Biol. Chem. 277, 6273-6279.

7.

Hung, Y. T., Lin, M. S., Chen, W. Y., Wang, S. S. (2010) Investigating the effects of sodium dodecyl sulfate on the aggregative behavior of hen egg-white lysozyme at acidic pH, Colloids. Surf B. Biointerfaces 81, 141-151.

8.

Cappai,

R., Leck,

S. L., Tew, D. J., Williamson,

N. A., Smith,

D. P., Galatis,

D., Sharples, R. A., Curtain, C. C., Ali, F. E., Cherny, R. A., Culvenor, J. G., Bottomley, 36


Page 37 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

S. P., Masters, C. L., Barnham, K. J., Hill, A. F. (2005) Dopamine promotes alphasynuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway, FASEB. J. 19, 1377-1379. 9.

Naidu, K.T., and Prabhu, N.P. (2011) Protein-surfactant interaction: sodium dodecyl sulfate-induced unfolding of ribonuclease A, J. Phys. Chem B. 115(49), 14760-14767.

10.

Otzen,

D.E.

(2002)

Protein unfolding in detergents: effect of micelle structure, ionic strength, pH, and temperature. Biophys. J. 83, 2219-2230. 11.

Thongngam,

M., McClements,

D.J.

(2005)

Influence

of pH,

ionic

strength,

and temperature on self-association and interactions of sodium dodecyl sulfate in the absence and presence of chitosan. Langmuir. 21, 79-86. 12.

Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F., Naiki, H. (2004) Low concentrations of sodium dodecyl sulfate induce the extension of beta 2-microglobulin-related amyloid fibrils at a neutral pH, Biochemistry, 43, 1107511082.

13.

Shao, H., Jao, S., Ma, K., Zagorski, M. G. (1999) Solution structures of micelle-bound amyloid beta-(1-40) and beta-(1-42) peptides of Alzheimer's disease, J. Mol. Biol. 285, 755-773.

14.

Yamamoto, N., Hasegawa, K., Matsuzaki, K., Naiki, H., Yanagisawa, K. (2004) Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid betaprotein, J. Neurochem. 90, 62-69.

15.

Ferreon, A. C., and Deniz, A. A. (2007) Alpha-synuclein multistate folding thermodynamics: implications for protein misfolding and aggregation, Biochemistry 46, 4499-4509.

16.

Andersen, K. K., Oliveira, C. L., Larsen, K. L., Poulsen, F. M., Callisen, T. H., Westh, P., Pedersen,

J.

S., Otzen,

D.

(2009)

The role of decorated SDS micelles in sub-

CMC protein denaturation and association. J. Mol. Biol. 391, 207-226. 17.

Gudiksen, K. L., Gitlin, I., Moustakas, D. T., Whitesides, G. M. (2006) Increasing the net charge and decreasing the hydrophobicity of bovine carbonic anhydrase decrease the rate of denaturation with sodium dodecyl sulfate. Biophysical J. 91, 298-310. 37


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18.

Page 38 of 55

Lee, A., Tang, S. K., Mace, C. R., Whitesides, G. M. (2011) Deanaturation of proteins by SDS and tetraalkylammonium dodecyl sulfates, Langmuir. 27, 11560-11574.

19.

Chiti, F., and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75, 333-366.

20.

Yutani, K., Takayama, G., Goda, S., Yamagata, Y., Maki, S., Namba, K., Tsunasawa, S., Ogasahara, K. (2000) The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus, Biochemistry 39, 27692777.

21.

Kurouski, D., Luo, H., Sereda, V., Robb, F. T., Lednev, I. K. (2012) Rapid degradation kinetics of amyloid fibrils under mild conditions by an archaeal chaperonin, Biochem. Biophys. Res. Commun. 422, 97-102.

22.

Melo, A. M., Loura, L. M., Fernandes, F., Villalaín, J., Prieto, M., Coutinho, A. (2014) Electrostatically driven lipid-lysozyme mixed fibers display a multilamellar structure without amyloid features, Soft Matter 10, 840-850.

23.

Khan, J. M., Qadeer, A., Chaturvedi, S. K., Ahmad,

E., Rehman, S. A., Gourinath,

S., Khan, R. H. (2012) SDS can be utilized as an amyloid inducer: a case study on diverse proteins, PLoS One 7(1), e29694. 24.

Kapoor. D., Singh, B., Karthikeyan, S., and Guptasarma, P. (2010) A functional comparison of the TET aminopeptidases of P. furiosus and B. subtilis with a proteinengineered variant recombining the former”s structure with the latter”s active site. Enzyme. and Microbial. Technology. 46, 1–8.

25.

Prakash, S., Sundd, M., and Guptasarma, P. (2014) The key to the extraordinary thermal stability of P. furiosus holo-rubredoxin: iron binding-guided packing of a core aromatic cluster responsible for high kinetic stability of the native structure, PLoS One 9(3), e89703.

26.

Sharma, P., and Guptasarma, P. (2015) Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli, Biochem. Biophys. Res. Commun. 460, 753-758.

27.

Lakowicz,

J.

R.

(1999)

Principles

of

Fluorescence

Academic/Plenum Press, New York. 38


Spectroscopy,

Kluwer

Page 39 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

28.

Wadai, H., Yamaguchi, K., Takahashi, S., Kanno, T., Kawai, T., Naiki, H., Goto, Y. (2005) Stereospecific amyloid-like fibril formation by a peptide fragment of beta2microglobulin. Biochemistry. 44,157-164..

29.

Wu, C., Wang, Z., Lei, H., Duan, Y., Bowers, M. T., Shea, J. E. (2008) The binding of thioflavin T and its neutral analog BTA-1 to protofibrils of the Alzheimer's disease Abeta(16-22) peptide probed by molecular dynamics simulations, J. Mol. Biol. 384, 718729.

30.

Ookoshi,

T., Hasegawa, K., Ohhashi, Y., Kimura, H., Takahashi, N., Yoshida,

H., Miyazaki, R., Goto, Y., Naiki, H. (2008) Lysophospholipids induce the nucleation and extension of beta2-microglobulin-related amyloid fibrils at a neutral pH, Nephrol. Dial. Transplant. 23, 3247-3255. 31.

Vilasi, S., Sarcina, R., Maritato, R., De Simone, A., Irace, G., Sirangelo, I. (2011) Heparin induces harmless fibril formation in amyloidogenic W7FW14F apomyoglobin an d amyloid aggregation inwild-type protein in vitro. PLoS One. 6, e22076.

32.

Motamedi-Shad,

N., Garfagnini,

T., Penco,

A., Relini,

A., Fogolari,

F., Corazza,

A., Esposito, G., Bemporad, F., Chiti, F. (2012) Rapid oligomer formation of human muscle acylophosphatase induced by heparin sulfate. Nat. Struct. Mol. Biol. 19, 547-554. 33.

Jain, S., Udganokar, J. B. (2008) Evidence for stepwise formation of amyloid fibril by mouse prion protein. J. Mol. Biol. 382, 1228-1241.

34.

Kumar, S., Udgankar, J.B. (2009) Conformational conversion may procede or follow aggregate elongation on alternative pathways of amyloidprotofibril formation. J. Mol. Biol. 385, 1266-1276.

35.

Roterman, I., KrUI, M., Nowak, M., Konieczny, L., Rybarska, J., Stopa, B., Piekarska, B., Zemanek, G. (2001) Why Congo red binding is specific for amyloid proteins - model studies and a computer analysis approach, Med. Sci. Monit. 7, 771-784.

36.

Millucci, L., Raggiaschi, R., Franceschini, D., Terstappen, G., Santucci, A. (2009) Rapid aggregation and assembly in aqueous solution of A beta (25-35) peptide, J. Biosci. 34, 293-303.

37.

Millucci, L., Raggiaschi, R., Franceschini, D., Terstappen, G., Santucci, A. (2003) Nucleation-dependent conformational conversion of the Y145Stop variant of human 39


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 55

prion protein: structural clues for prion propagation, Proc. Natl. Acad. Sci. U. S. A. 100, 12069-12074. 38.

Smith, D. P., Jones, S., Serpell, L. C., Sunde, M., Radford, S. E. (2003) A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation, J. Mol. Biol. 330, 943-954.

39.

Singh, J., Sabareesan, A. T., Mathew, M. K., Udgaonkar, J. B. (2012) Development of the structural core and of conformational heterogeneity during the conversion of oligomers of the mouse prion protein to worm-like amyloid fibrils, J. Mol. Biol. 423, 217-231.

40.

Gosal, W. S., Morten, I. J., Hewitt, E. W., Smith, D. A., Thomson, N. H., Radford, S. E. (2005) Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid, J. Mol. Biol. 351, 850-864.

41.

Hugonin, L., Barth, A., Gräslund, A., Perálvarez-Marín, A. (2008) Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate, Biochim. Biophys. Acta. 1778, 2580-2587.

42.

Saunders, H. M., Hughes, V. A., Cappai, R., Bottomley, S. P. (2013) Conformational behavior and aggregation of ataxin-3 in SDS, PLoS One 8(7), e69416.

43.

Khan, J. M., Abdulrehman, S. A., Zaidi,

F. K., Gourinath, S., Khan, R. H. (2014)

Hydrophobicity alone can not trigger aggregation in protonated mammalian serum albumins, Phys. Chem. Chem. Phys. 16, 5150-5161. 44.

Qadeer, A., Ahmad, E., Zaman, M., Khan, M. W., Khan, J. M., Rabbani, G., Tarique, K. F., Sharma, G., Gourinath, S., Nadeem, S., Badr, G., Khan, R. H. (2013) Concentrationdependent antagonistic persuasion of SDS and naphthalene derivatives on the fibrillation of stem bromelain, Arch. Biochem. Biophys. 540, 101-116.

45. Necula, M., Chirita, C. N. & Kuret, J. (2003) Rapid anionic micelle-mediated alphasynuclein fibrillization in vitro. J. Biol. Chem. 278, 46674–46680. 46.

Giehm, L., Oliveira, C.L., Christiansen, G., Pedersen, J.S., Otzen, D.E. (2010) SDSinduced fibrillation of alpha-synuclein:

an alternative fibrillation pathway.

J

Mol

Biol. 401, 115-133. 47.

Ahmad, M. F., Raman, R.T., Rao, ChM. R. (2006) Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human alpha-synuclein, J. Mol. Biol. 364, 1061-1072. 40


Page 41 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

48.

Sureshbabu, N., Kirubagaran, R., and Jayakumar, R. (2009) Surfactant-induced conformational transition of amyloid beta-peptide, Eur. Biophys. J. 38, 355-367.

49.

Khan, J. M., Chaturvedi, S. K., Rahman, S. K., Ishtikhar,

M., Qadeer,

A., Ahmad,

E., Khan, R. H. (2014) Protonation favors aggregation of lysozyme with SDS, Soft Matter 10, 2591-2599.

Figure Legends Figure 1: Panel A: Turbidity in solutions of hyperthermophile proteins (5 µM) monitored as changes in absorbance at 350 nm. PfuTIM (-■-), PfuAmy (-●-), PfRd (-▲-), PfuASL (-▼-) and PfuAP (-♦-)incubated at different concentrations of SDS in buffers of different pH : PfuTIM (pH 4.5), PfuAmy (pH 3.6), PfRd (pH 3.0), PfuASL (pH 3.5) and PfuAP (pH 4.1). Panel B: Proteins (5 µM) incubated at different concentration of SDS at pH 7.4, as a control experiment. Panel C: Trp fluorescence anisotropy measurements of proteins (5 µM) at different SDS concentrations, at pH values two units below pI. Panel D: Fluorescence intensity of ThT (5 µM) allowed to bind to proteins (5 µM), monitored at 485 nm and plotted for different SDS concentrations. Prior to 41


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 55

measurements all samples were incubated overnight, in buffers of 20 mM strength, at suitable pH values (two units below/above pI) and stirred to resuspend any sedimented protein.

Figure 2: Kinetics of SDS-induced amyloid fibril formation, measured by monitoring increase in ThT fluorescence with time. Panel A: PfuAmy. Panel B: PfuAP. Panel C: PfuASL. Panel D: PfRd. Panel E: PfuTIM. The blue trace shows ThT fluorescence at a pH two units below the pI, in the absence of SDS. The black trace shows ThT fluorescence at a pH two units below the pI in the presence of 1 mM SDS. The red trace shows ThT fluorescence under neutral pH conditions (pH 7.4) in the presence of 1 mM SDS. ThT was added immediately before measurement commenced. Figure 3: Congo Red (CR, 5 µM) binding to aggregates of the five hyperthermophile proteins from Pyrococcus furiosus, monitored through measurement of absorbance of CR as a function of wavelength. Traces are shown for soluble protein in the presence of CR at physiological pH (filled squares) and for protein aggregated in the presence of 1 mM SDS at a pH two units lower than the protein’s pI, sedimented, and resuspended, back to identical volume and concentration (5µM), in buffer of physiological pH before being allowed to interact with CR (open circles) . Panel A: PfuAmy. Panel B: PfuAP. Panel C: PfuASL. Panel D: PfRd. Panel E: PfuTIM. Panel F: Control CR spectra of CR alone, at pH 7.0, in the absence of any protein or SDS (filled squares), and in the presence of 1 mM SDS (open circles). Congo Red was added 30 min before measurement, and incubation with the dye was performed in the dark.

Figure 4: Representative AFM scans of hyperthermophile proteins, revealing formation of amyloid fibrils following incubation with SDS (1 mM) in buffer of pH two units below the pI. 42


Page 43 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Height data for the fibrils was measured (shown alongside, in separate adjoining panels) along a line trajectory shown in the AFM image as a horizontal black line. Panel A: PfuAP (overnight incubation). Panel B: PfuASL (overnight incubation). Panel C: PfuASL (30 min incubation). Panel D: PfuASL (30 min incubation; 3D view). Also see Supplementary Figure S2 for AFM images obtained after 10 min incubation.

Figure 5: Far-UV CD spectra of the five hyperthermophile proteins (5µM) following overnight incubation with SDS. Values of SDS concentrations and buffer pH are mentioned as inset. Panel A: PfuAmy. Panel B: PfuAP. Panel C: PfuASL. Panel D: PfRd. Panel E: PfuTIM.

Figure 6: Panel A: Turbidity in solutions of T. maritima PEPM50 (5 µM) monitored as absorbance at 350 nm. PEPM50 was incubated at different concentrations of SDS in buffers of different pH as given alongside (pH=4 --, pH=5 -Ο-, pH=7 -∆-). Panel B: Fluorescence intensity of ThT (5 µM) allowed to bind to the PEPM50 (5 µM), monitored at 485 nm and plotted for different SDS concentrations. Prior to measurements all samples were incubated overnight, in buffers of 20 mM ionic strength, at suitable pH values. Panel C: Far-UV CD spectra of PEPM50 (5µM) following overnight incubation with SDS. Values of SDS concentrations and buffer pH are mentioned as inset.

Figure 7 : Two dimensional PAGE images showing PfuAMY and PfRd (panel A), PfuASL (panel B), PfuAP (panel C), PfuTIM (panel D), and PEPM50 (panel E). Molecular weight and pI ranges are shown.

43


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8: A schematic diagram visually depicting the differential availabilities of free SDS molecules versus SDS in micellar form, at concentrations just below, and just above, the critical micellar concentration (CMC). The two upper panels depict the shift in equilibrium from free SDS to micellar SDS. The two lower panels depict the consequences of micelle formation on the interaction of SDS with proteins, emphazing the point that from purely geometric and steric points of view SDS molecules on the ‘far-side’ of a micelle are not available to interact with positive charges on protein surfaces.

Figure 9: A schematic diagram visually depicting the differential conformational outcomes of exposure of proteins to mixtures of free SDS and micelles. When micelles are in a minority (or absent) at low SDS concentrations, more interactions of proteins occur with free SDS molecules and individual SDS molecules bind to positively-charged residues on the surfaces of proteins in native conformation, leading to conversion of conformation into an aggregation-prone, amyloidforming state characterized by a negative ~330 nm CD band, irrespective of the structure of the native molecule (i.e., whether helical, or beta sheet, or a combination thereof). In contrast, when micelles are in a majority, and there are too few individual SDS molecules present, proteins adopt some other non-native conformation which could be a helical conformation, as reported for many proteins (and shown in this paper), or some other non-native conformation, including denatured randomly-coiled conformation.

44


Page 44 of 55

Page 45 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

FOR TABLE OF CONTENTS USE ONLY Table of Contents (TOC) Graphic

45


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1: Panel A: Turbidity in solutions of hyperthermophile proteins (5 µM) monitored as changes in absorbance at 350 nm. PfuTIM (-■-), PfuAmy (-●-), PfRd (-▲-), PfuASL (-▼-) and PfuAP (-♦-)incubated at different concentrations of SDS in buffers of different pH : PfuTIM (pH 4.5), PfuAmy (pH 3.6), PfRd (pH 3.0), PfuASL (pH 3.5) and PfuAP (pH 4.1). Panel B: Proteins (5 µM) incubated at different concentration of SDS at pH 7.4, as a control experiment. Panel C: Trp fluorescence anisotropy measurements of proteins (5 µM) at different SDS concentrations, at pH values two units below pI. Panel D: Fluorescence intensity of ThT (5 µM) allowed to bind to proteins (5 µM), monitored at 485 nm and plotted for different SDS concentrations. Prior to measurements all samples were incubated overnight, in buffers of 20 mM strength, at suitable pH values (two units below/above pI) and stirred to resuspend any sedimented protein. 254x190mm (96 x 96 DPI)


Page 46 of 55

Page 47 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2: Kinetics of SDS-induced amyloid fibril formation, measured by monitoring increase in ThT fluorescence with time. Panel A: PfuAmy. Panel B: PfuAP. Panel C: PfuASL. Panel D: PfRd. Panel E: PfuTIM. The blue trace shows ThT fluorescence at a pH two units below the pI, in the absence of SDS. The black trace shows ThT fluorescence at a pH two units below the pI in the presence of 1 mM SDS. The red trace shows ThT fluorescence under neutral pH conditions (pH 7.4) in the presence of 1 mM SDS. ThT was added immediately before measurement commenced. 186x235mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3: Congo Red (CR, 5 µM) binding to aggregates of the five hyperthermophile proteins from Pyrococcus furiosus, monitored through measurement of absorbance of CR as a function of wavelength. Traces are shown for soluble protein in the presence of CR at physiological pH (filled squares) and for protein aggregated in the presence of 1 mM SDS at a pH two units lower than the protein’s pI, sedimented, and resuspended, back to identical volume and concentration (5µM), in buffer of physiological pH before being allowed to interact with CR (open circles) . Panel A: PfuAmy. Panel B: PfuAP. Panel C: PfuASL. Panel D: PfRd. Panel E: PfuTIM. Panel F: Control CR spectra of CR alone, at pH 7.0, in the absence of any protein or SDS (filled squares), and in the presence of 1 mM SDS (open circles). Congo Red was added 30 min before measurement, and incubation with the dye was performed in the dark. 279x361mm (300 x 300 DPI)


Page 48 of 55

Page 49 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4: Representative AFM scans of hyperthermophile proteins, revealing formation of amyloid fibrils following incubation with SDS (1 mM) in buffer of pH two units below the pI. Height data for the fibrils was measured (shown alongside, in separate adjoining panels) along a line trajectory shown in the AFM image as a horizontal black line. Panel A: PfuAP (overnight incubation). Panel B: PfuASL (overnight incubation). Panel C: PfuASL (30 min incubation). Panel D: PfuASL (30 min incubation; 3D view). Also see Supplementary Figure S2 for AFM images obtained after 10 min incubation. 215x279mm (150 x 150 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5: Far-UV CD spectra of the five hyperthermophile proteins (5µM) following overnight incubation with SDS. Values of SDS concentrations and buffer pH are mentioned as inset. Panel A: PfuAmy. Panel B: PfuAP. Panel C: PfuASL. Panel D: PfRd. Panel E: PfuTIM. 190x254mm (96 x 96 DPI)


Page 50 of 55

Page 51 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 6: Panel A: Turbidity in solutions of T. maritima PEPM50 (5 µM) monitored as absorbance at 350 nm. PEPM50 was incubated at different concentrations of SDS in buffers of different pH as given alongside (pH=4 -ν-, pH=5 -Ο-, pH=7 -∆-). Panel B: Fluorescence intensity of ThT (5 µM) allowed to bind to the PEPM50 (5 µM), monitored at 485 nm and plotted for different SDS concentrations. Prior to measurements all samples were incubated overnight, in buffers of 20 mM ionic strength, at suitable pH values. Panel C: Far-UV CD spectra of PEPM50 (5µM) following overnight incubation with SDS. Values of SDS concentrations and buffer pH are mentioned as inset. 210x297mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Page 52 of 55

Page 53 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 8: A schematic diagram visually depicting the differential availabilities of free SDS molecules versus SDS in micellar form, at concentrations just below, and just above, the critical micellar concentration (CMC). The two upper panels depict the shift in equilibrium from free SDS to micellar SDS. The two lower panels depict the consequences of micelle formation on the interaction of SDS with proteins, emphazing the point that from purely geometric and steric points of view SDS molecules on the ‘far-side’ of a micelle are not available to interact with positive charges on protein surfaces. 161x103mm (96 x 96 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9: A schematic diagram visually depicting the differential conformational outcomes of exposure of proteins to mixtures of free SDS and micelles. When micelles are in a minority (or absent) at low SDS concentrations, more interactions of proteins occur with free SDS molecules and individual SDS molecules bind to positively-charged residues on the surfaces of proteins in native conformation, leading to conversion of conformation into an aggregation-prone, amyloid-forming state characterized by a negative ~330 nm CD band, irrespective of the structure of the native molecule (i.e., whether helical, or beta sheet, or a combination thereof). In contrast, when micelles are in a majority, and there are too few individual SDS molecules present, proteins adopt some other non-native conformation which could be a helical conformation, as reported for many proteins (and shown in this paper), or some other non-native conformation, including denatured randomly-coiled conformation. 254x190mm (96 x 96 DPI)


Page 54 of 55

Page 55 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

FOR TABLE OF CONTENTS USE ONLY - TOC GRAPHIC 58x37mm (96 x 96 DPI)


The Achilles' Heel of âUltrastableâ Hyperthermophile Proteins

Recommend Documents

The Achilles' Heel of âUltrastableâ Hyperthermophile Proteins