Calorimetric and Spectroscopic Studies of the Effects of the Cell

Oct 17, 2017 - Department of Physics and Computer Science, Xavier University of ... The objective of this study is to measure and compare the effects ...
1 downloads 0 Views 3MB Size
Subscriber access provided by University of Florida | Smathers Libraries

Article

Calorimetric and spectroscopic studies of the effects of the cell penetrating peptide Pep-1 and the antimicrobial peptide combi-2 on vesicles of mimicking Escherichia coli membrane Nsoki Phambu, Bashiyar Almarwani, Amjad Alwadai, Esther Nzuzi Phambu, Natalie Faciane, Carmel Marion, and Anderson Sunda-Meya Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01910 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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.

Langmuir 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 32

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

Langmuir

Calorimetric and spectroscopic studies of the effects of the cell penetrating peptide Pep-1 and the antimicrobial peptide combi-2 on vesicles of membranes mimicking Escherichia coli 82x44mm (300 x 300 DPI)

ACS Paragon Plus Environment

Langmuir

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

Calorimetric and spectroscopic studies of the effects of the cell penetrating peptide Pep-1 and the antimicrobial peptide combi-2 on vesicles mimicking Escherichia coli membrane. Nsoki Phambu1*, Bashiyar Almarwani 1, Amjad Alwadai 1, Esther N. Phambu2, Natalie Faciane3, Carmel Marion3, and Anderson Sunda-Meya3* 1

2

Department of Chemistry, Tennessee State University, Nashville, TN 37209, USA

Department of Chemical & Biomolecular Engineering, New York University, Brooklyn, NY 11201, USA

2

Department of Physics and Computer Science, Xavier University of Louisiana, New Orleans, LA 70125, USA

Corresponding Authors *Anderson Sunda-Meya, [email protected] * Nsoki Phambu, [email protected]; KEYWORDS 1

ACS Paragon Plus Environment

Page 2 of 32

Page 3 of 32

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

Langmuir

Cell-Penetrating Peptides; Antimicrobial Peptides; Peptide Self-Assembly; Peptide-Lipid Interactions; Conformation; TGA; DSC; SEM

ABSTRACT

The objective of this study is to measure and compare the effects of the cell penetrating peptide (CPP) Pep-1 and the antimicrobial peptide (AMP) combi-2 on vesicles of membranes mimicking Escherichia coli (E. coli). To characterize the effects of Pep-1 and combi-2 on E. coli membrane vesicles, a combination of five biophysical techniques was employed: fluorescence, infrared, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Upon addition of E. coli membranes, tryptophan fluorescence intensity of Pep-1 showed a sudden blue shift and decreased in a non-concentrationdependent manner while the intensity of combi-2 decreased in a concentration-dependent manner, most significantly for a very low peptide-to-lipid ratio of 1:40. Complexes of Pep-1 and combi-2 with E. coli membrane mimicking vesicles having shown a significant blue shift in fluorescence intensity were then prepared and studies in freeze-dried states. IR results indicate that Pep-1 and combi-2 adopt a major 310-helix structure in the presence of E. coli membrane mimicking vesicles at low peptide concentration. Pep-1 and combi-2 have a similar effect on E. coli membrane mimicking vesicles at low concentration even though combi-2 is in the interfacial region of the bilayer while Pep-1 is located between the interfacial region and the hydrophobic region. Combi-2 at low concentration acts as a CPP. TGA and DSC results reveal that combi-2 has a stabilizing effect on E. coli at any concentration while Pep-1 stabilizes the E. coli membrane only at high concentration. Both peptides show a preferential interaction with one of the anionic lipids leading to clustering in E. coli membrane. SEM images reveal that Pep-1 and 2

ACS Paragon Plus Environment

Langmuir

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

combi-2 form superstructures including fibrils in the presence of E. coli membrane mimicking vesicles. Calorimetric and spectroscopic techniques may be used in a complementary way with imaging techniques to gain more insights into peptide-lipid interactions. INTRODUCTION Short length peptides have recently gained growing attention for biomedical applications. Among these peptides, there are cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs). The research field of cell-penetrating peptides (CPPs), or alternatively called protein transduction domains (PTDs), has increased rapidly in the last few years1–4. CPPs constitute one of the most promising tools for the delivery of biopharmaceuticals into cells and therefore play a key role in future development of therapeutics1, but their mechanism of entry into the cell is still unclear1,3–5. One of the most used CPPs is Pep-1, a short amphipathic peptide of 21 amino acid residues consisting of a hydrophobic tryptophan-rich domain and a hydrophilic lysine-rich domain separated by a spacer1,5. AMPs have the potential to be developed as antibiotics, but their killing mechanism is still open to debate6. Combi-2 is an AMP found through combinatorial peptide chemistry7; it is a tryptophan and arginine-rich AMP8–10. It has been shown to be effective against Gram-positive and Gram-negative bacteria. Very few studies have investigated the interaction of combi-2 with model membrane mimicking vesicles. Combi-2 contains a phenylalanine residue in addition of tryptophan and arginine residues. Because of their amphiphilicity, the AMPs are found to directly interact with a cell membrane, making the target cell die6,11. More interestingly, AMPs are also found to exhibit high cell-type selectivity, that is, they can kill the invading cells but leave the host cells unharmed. At present, the factors which determine the selectivity are not all well understood12. From a mechanistic point of view, potentially all CPPs are AMPs and all 3

ACS Paragon Plus Environment

Page 4 of 32

Page 5 of 32

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

Langmuir

AMPs are CPPs, and there is no reason to study these two classes separately11. Ding et al.3 investigated molecular interactions between Pep-1 and simple model cell membrane mimicking vesicles. They highlighted two issues. First, the concentration of CPPs required for translocation to occur is usually lower than that required for AMPs to disrupt the cell membranes and, therefore, a technique with high sensitivity to study CPPs is necessary. Second, studies on the interactions of CPPs with more advanced model cell membrane systems are required to fully understand the behavior of CPPs. The objective of this study is to address those issues by using techniques with high sensitivity and by studying the interactions of Pep-1 and combi-2 with more advanced model membranes. We decided to measure and compare the effects of Pep-1 and combi-2 on vesicles of membranes mimicking E. coli using fluorescence, infrared, TGA, DSC and SEM. We chose Pep-1 and combi-2 because they are similar: both have at least one arginine and contain hydrophilic residues. Pep-1 contains several lysine residues while combi-2 contains arginine residue. In addition, combi-2 contains a phenylalanine residue. Vogel et al.7 suggested that the bactericidal action of the peptide combi-2 may involve translocation across the membrane. Finally, in-depth physicochemical investigations on the behavior of CPPs and AMPs are rare11 and studies using imaging of vesicles are scarce1–3. E. coli is a well-studied bacterium12,13. The lipid composition of E. coli’s membrane was opened to debate for a long time6,14–16. However, there seems to be a consensus on the composition

of

E.

coli

membrane

mimicking

vesicles.

It

contains

~75%

phosphatidylethanolamine (PE), ~20% phosphatidylglycerol (PG) and ~5% cardiolipin (CL). The latter two lipids are negatively charged, while PE is neutral. Here we report on the perturbations of two tryptophan-rich peptides Pep-1 and combi-2 on bilayers of a very 4

ACS Paragon Plus Environment

Langmuir

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

challenging model membrane that is a ternary mixture of lipids mimicking E. coli membrane mimicking vesicles. The interaction was first performed in a wide range of peptide-to-lipid molar ratios in liquid to find the most interesting peptide-to-lipid molar ratios. And then solid samples prepared from the most interesting peptide-to-lipid molar ratios found from the fluorescence results were investigated in freeze-dried state. To characterize the effects of Pep-1and combi-2 on E. coli membrane vesicles, we chose fluorescence, infrared, SEM, TGA and DSC. Fluorescence spectroscopy has been extensively used to investigate peptide-membrane interactions in liquid phase12,17,18. The changes in some of the intrinsic fluorescence parameters of peptides containing fluorescent amino acid residues can be easily used to measure the insertion of a peptide on a membrane, without the need of peptide labeling. Additionally, the fluorescent amino acid residues of a protein can also be used as energy transfer donors, enabling the quantification of the extension even of the weak interactions associated with most of the membrane adsorption processes12,17. In addition to the fluorescence quantum yield and lifetime, the position of the maximum of the fluorescence spectrum of tryptophan residues, is also sensitive to its environment, and the transfer of the peptide from the aqueous (polar) environment to the lipid bilayer (less polar), characteristically causes a blueshift of emission from around 360 nm to as low as 310 nm19. This property can therefore be used to obtain a reasonable description of the location/environment of peptide’s tryptophan residues in the membrane12,18,20,21. Infrared spectroscopy has been used extensively to study the secondary structure of peptides in the presence and absence of membranes1,22–25. The amide I group of peptides gives the secondary structures of peptides. Because the amide I bands are usually too broad, a decomposition treatment is often used to determine the content of each secondary structure of the peptide1. 5

ACS Paragon Plus Environment

Page 6 of 32

Page 7 of 32

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

Langmuir

TGA and DSC are rarely used to study peptides in the solid state. More often, TGA and DSC are used to study the aggregation and phase transitions of peptides in the 20-80oC temperature range2,11,26–29. Our system is a ternary lipid mixture composed of DPPE, DPPG and cardiolipin. Since Pep-1 and combi-2 are positively charged, it is important to evaluate the possible lipid recruitment induced by the peptide11. In addition, due to the fact that cardiolipin has no observable transition between 0 and 100 oC11,30, we decided to use simultaneous TGA and DSC techniques on freeze-dried samples up to 700oC to observe the breaking of the bonds31–36. Peptide-lipid interactions have been investigated using imaging techniques such as transmission electron microscopy (TEM) and scanning electron microcopy (SEM), atomic force microscopy (AFM)37–39. The main advantage of these techniques is the direct visualization of the effect of the peptide addition or adsorption to the lipid layer or cell wall. In this study, we use SEM technique to observe the morphological changes induced by the peptide on the E. coli membrane. EXPERIMENTAL Materials Dimyristoylphosphotidylethanolamine

(DPPE),

Dimyristoylglycerophosphorylglycerol

(DPPG), and cardiolipin (CL) were purchased from Avanti Polar Lipid Inc. and used without further purification. The peptides Pep-1 (KETWWETWWTEWSQPKKKRKV) and combi-2 (FRWWHR) were obtained from GenScript (>96 % purity) and used as received. Sample and vesiclePreparation. The mixture of lipids used to mimic E coli membrane composition is that used by Wei-Chin et al.40, which is a three-component system CL/DPPG/DPPE (1:5:15 molar ratio). The solid phases were obtained for the following sample compositions: Pep-1 with the lipid mixture Pep6

ACS Paragon Plus Environment

Langmuir

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

1/CL/DPPG/DPPE (1:1:5:15 molar ratio) is called Pep-1/lipid [1:1], Pep-1/CL/DPPG/DPPE (0.025:1:5:15 molar ratio) is called Pep-1/lipid

[1:40], combi-2/CL/DPPG/DPPE (1:1:5:15

molar ratio) is called combi-2/lipid [1;1], and combi-2/CL/DPPG/DPPE (0.025:1:5:15 molar ratio) is called combi-2/lipid [1:40]. Vesicles were prepared using pure CL, DPPG, and DPPE powders. The appropriate amount of dried lipid was weighed and dissolved in chloroform and vortex for 5 minutes. The sample was dried under a stream of nitrogen gas for 6 hours and under vacuum overnight. A thin lipid film was formed on the wall of the vial. The thin lipid film was then hydrated with water to a total lipid concentration of 10 µM. Small Unilamellar Vesicles (SUVs) were prepared by sonication of the milky lipid suspension using a Sonic Dismembrator Ultrasonic Processor (Model FB-50 including a standard 1/8” diameter microtip, in titanium alloy) from Fisher Scientific for about an hour in an ice bath until the solution became transparent. Stock solutions of Pep-1 and combi2 with a defined concentration were also prepared. For peptide binding experiments, an appropriate volume of either Pep-1 or combi-2 was mixed to an appropriate volume of E. coli vesicles to make up the different solutions with different proportions of E. coli vesicles. Preparation of solid phases: we prepared solid samples of Pep-1 and combi-2 and their complexes with E. coli vesicles by drying a certain volume of a solution of interest. A vacuum apparatus FreeZone 6 from LABCONCO was used to obtain the freeze-dried samples. Characterization. Fluorescence spectroscopy. The ability of Pep-1 and combi-2 peptides to self-aggregate in aqueous solutions was monitored by tryptophan fluorescence. The excitation wavelength was set at 280 nm and the emission was scanned from 290 to 490 nm with a slit width of 5 nm for both peptides12,19,28. First, peptides were titrated in the concentration range of 10-3to 10-7 M. Peptides 7

ACS Paragon Plus Environment

Page 8 of 32

Page 9 of 32

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

Langmuir

at the concentration range 10-5 to 10-7 M showed no aggregation in solution phase, so the peptide concentration of 10-5 M was chosen. Peptide-lipid interaction was studied by monitoring the changes in tryptophan fluorescence emission spectra upon addition of increasing amount of lipid12,19,20. The change in the intensity and shift in the wavelength was monitored at different peptide-to-lipid concentration ratios. Measured intensities were normalized using an internal standard. The wavelength with the maximum fluorescence emission was then plotted against the lipid concentration12. Because combi-2 contains in addition a phenylalanine residue, phenylalanine fluorescence was also monitored using the excitation wavelength of 260 nm. Infrared spectroscopy. Infrared spectra were obtained using a Fourier Transform instrument (Thermo Scientific iS10) equipped with a single reflection Ge attenuated total reflectance accessory. Solid samples were used. The change in vibration modes of peptides with and without the lipid mixture was then monitored. The spectra were recorded at room temperature between 4000 and 600 cm-1 at 4 cm-1 resolution and 64 scans were accumulated. Routine smoothing and normalization were applied to all the infrared spectra25,41. Determination of the secondary structure. The determination of the secondary structure from infrared spectra shows a matter of concern41. This is because the bands characteristic of amide I vibration are broad. A curve fitting treatment was carried out to estimate quantitatively the relative proportion of each component representing each type of secondary structure. FTIR spectra of peptides with and without lipid were recorded and used in this study1,41. First, the FTIR spectra were normalized. Then, a linear baseline was used between 1700 and 1600 cm-1, and a FT self-deconvolution was applied. The frequencies, the number of peaks to be fitted and the half width (15 cm-1) of each peak to start a least square iterative curve fitting procedure, were obtained from the second derivative of the original FTIR spectra. Then the deconvolution of the 8

ACS Paragon Plus Environment

Langmuir

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 10 of 32

amide I band was performed with OMNIC Software (Thermo Fisher Scientific, Waltham, MA), and analyzed as a sum of Gaussian curves, with consecutive optimization of amplitudes, band positions, half-widths, and Gaussian composition of individual bands. The amount of each secondary structure element is given in percentage terms, by dividing the area of one amide I band component by the area of the sum of all amide band component areas. We assigned the types of secondary structures based on the data found in the literature1,41–43. Thermogravimetric analysis (TGA) and Differential Scanning calorimetry (DSC). Thermal stability and phase transitions of peptides and their complexes were recorded by Thermogravimetric Analysis (TGA)26–28 and Differential Scanning Calorimetry (DSC)44–47 with a LINSEIS STA PT1600 instrument. This instrument determines simultaneous changes (in a single run) of mass and caloric reactions of a sample. The instrument performs tests from ultrahigh vacuum 10-4 mbar to 5 bar over pressure. Samples weighing 4-6 mg were put in an aluminum pan and an empty pan was used as a reference. Investigations were performed between room temperature and 700oC with a heating rate of 10oC per minute. At this heating rate of 10oC per minute, the degradation of the samples was reduced. Transition parameters were obtained with the manufacturer’s pre-installed software48,49. The aliquots from the same freezedried samples are used for infrared, SEM, TGA and DSC. Scanning electron microscope (SEM). Scanning electron microscopy (SEM). The SEM images were obtained in a S4800 field emission scanning electron microscope (SEM) (Hitachi High Technologies America Inc., Gaithersburg, MD). All samples were gold sputter-coated at 2.0 mA for 1 min (K550X Sputter Coater, Quorum Technologies Ltd., West Sussex, UK). An accelerating voltage of 5 kV was used for all SEM observations. RESULTS AND DISCUSSION 9

ACS Paragon Plus Environment

Page 11 of 32

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

Langmuir

Fluorescence spectroscopy The membrane response to the peptide’s activity is revealed by the change of fluorescence emission intensity of peptide as a function of peptide-to-lipid molar ratio. At the excitation wavelength of 280 nm, the emission is due to tryptophan residue, and its interaction with the lipidic system can be appreciated from the spectral shifts observed12,17,18. The blue shifts in tryptophan emission maxima of Pep-1 and combi-2 in E. coli membrane mimetic vesicles are given in figure 1. The fluorescence emission spectra of Pep-1 and combi-2 peptides under our experimental conditions are shown in figures S1 and S2, respectively, in the supplemental materials section. The fluorescence spectrum of Pep-1 in aqueous solution showed an emission maximum at 357 nm, which is expected for solvent exposed tryptophan residues7,17,20,21,47. The fluorescence emission maxima of Pep-1 exhibited both a sudden blueshift and a marked decrease in emission intensity in the presence of E coli lipid mixture when the lipid/ peptide ratio was increased from 0 to 40 (figure S1). To our knowledge, the average of 30 nm blueshift is shown for the first time. Deshayes et al.1 showed a blue shift of 22 nm when Pep-1 interacted with single vesicles of DOPC and DOPG. The subsequent shifts in emission wavelength of Pep-1 and combi-2 were plotted as a function of the peptide-to-lipid molar ratio (figure 1). The huge blue shift indicates that Pep-1 is deeply inserted in the hydrophobic core of phospholipid vesicles and the quenching of the emission intensity is consistent with a transfer between tryptophan residues47. The fluorescence spectrum of combi-2 in aqueous solution showed an emission maximum at 356 nm (figure S2). The fluorescence emission maxima of combi-2 exhibited a gradual decrease in emission intensity in the presence of E. coli lipid mixture when the peptide-to-lipid molar ratio was increased from 0 to 40 (table 1). A blue shift of 8 nm is observed only at the 1:40 peptide-to10

ACS Paragon Plus Environment

Langmuir

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 32

lipid molar ratio. The relatively small blue shift compared to Pep-1 indicates that combi-2 is not inserted deep into the hydrophobic core of phospholipid vesicles and the quenching of the emission intensity is consistent with a transfer between tryptophan residues. The phenylalanine emission fluorescence of combi-2 was also monitored using an excitation wavelength of 260 nm (results not shown). As expected, this non-polar residue did not show a significant change upon E. coli membrane addition12. But this hydrophobic residue may also prevent combi-2 from going deep into the hydrophobic core of the model membrane. Overall, Pep-1 lies deeper inside the hydrophobic core of the membrane than combi-2. In liquid phase, the incorporation of peptide into the membrane is not quantitative. A sample with the highest concentration of lipid may still contain a significant amount of peptide in water in such a way that the spectrum obtained does not correspond only to peptide incorporated into the membrane mimicking vesicles48. Based on the fluorescence data above, we chose to gain more insights into the complexes of E. coli membrane mimicking vesicles with the two peptides in solid phase. In line with the objective of this study, solid samples containing peptide-to-lipid molar ratios of 1:1 and 1:40 were prepared as described in the experimental section. The 1:1 molar ratio is commonly used because it represents equivalence. The 1:40 molar ratio gave a concomitant decrease in intensity and blue shift in frequency in both the fluorescence emission of Pep-1 and combi-2 in the E. coli membrane (figure 1). Structural analysis. FTIR has been widely used to investigate the secondary structure of peptides and proteins43,50– 54

. First the analysis of the original FTIR spectra of E. coli, Pep-1 and combi-2 with and without

E. coli membrane mimicking vesicles was done to observe how peptides affect E. coli membrane. Table 1 summarizes the wavenumbers characteristic of the main functional groups of 11

ACS Paragon Plus Environment

Page 13 of 32

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

Langmuir

these samples. From table 1, one can observe that the N-H band of E. coli at around 3374 cm-1 is significantly affected by the presence of peptides at any concentration. The NH group shifts to lower wavenumbers, indicating a strengthening of the H bonds. The presence of peptides does not significantly affect the acyl chain region of E. coli. This means that there is no ordering of the hydrocarbon chains when the peptides were bound. The FTIR spectrum of E. coli shows a peak at 1741 cm-1 assigned to the C=O band. This peak at such high wavenumber is assigned to non-hydrogen-bonded ester carbonyl groups22,42,55. In the presence of peptides, the peak shifts to a lower wavenumber at around 1738 cm-1, which indicates a dehydration of the lipid head group region but it is still assigned to non-hydrogen-bonded ester carbonyl groups24,42. The downshift indicates strengthening of the ester carbonyl group. From table 1, the IR spectrum of E. coli shows two intense peaks at 1223 and 1079 cm-1. The peaks at 1223 and 1079 cm-1 have been assigned to an asymmetric PO2- stretching mode (νas PO2-) and symmetric PO2-stretching modes (νs PO2-) of the phosphate group, respectively56. In the presence of Pep-1 and combi-2, the peaks are slightly lower in wavenumbers. The presence of peptides does not affect the phosphate group of E. coli. Subsequently, the secondary structure of Pep-1 and combi-2 with and without E. coli membrane mimicking vesicles was examined by comparison of FTIR spectra at amide I (17001600 cm-1) and amide II (1600-1500 cm-1) regions that are characteristic of peptides. The goal is to observe if the conformation of the peptides changes in the presence of E. coli membrane. Figure 2 presents the original FTIR spectra of the samples in the regions cited above. The FTIR spectrum of Pep-1 shows a broad dissymmetric peak with maximums at 1650 and 1625 cm-1, characteristic of helix and sheet structures, respectively. The FTIR spectrum of combi-2 presents a broad peak with a maximum at 1642 cm-1, suggesting the presence of random coils structures. 12

ACS Paragon Plus Environment

Langmuir

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 32

The FTIR spectra of the complexes are dominated by the strong C=O peak induced by E. coli. The amide bands in these regions are so broad that it is difficult to determine the secondary structure of the peptide. Since the amide I regions consist of broad bands, it was subject to curve-fitting in order to resolve the various subcomponents present in the amide I region43,53. The characteristic amide I peak described in the literature contains different secondary structures, including: strong intermolecular β-sheet (1622–1627 cm-1), strong intramolecular β-sheet (1628–1638 cm-1), weak β-sheet (1690–1703 cm-1), random coils (1637–1644 cm-1), α-helix (1645–1657 cm-1), 310-helix (1658–1666 cm-1), and turns (1668–1685 cm-1). The conformation ranges above are based on the experimental data and assignments collected from various authors and evaluated by our team43,51–54,57. The results of quantitative analysis of peptide conformation reveal that Pep-1 (figure S3) consists of a mixture of 310-helix (34%) and β-sheet (66%) structures. The β-sheet structures are characteristic of intermolecular H bonds with antiparallel orientation. Combi-2 adopts a major random coils structure (100%). Knowing that the secondary structures of peptides are different, we aimed to monitor if these differences translate to a different impact on membrane interfaces. In the presence of E. coli membrane mimicking vesicles, the contents of helix and sheet structures of Pep-1 are converted to 100% 310-helix structures at high peptide concentration and 100% β-sheets at low peptide concentration. The behavior of Pep-1 was expected as Pep-1 consisted mostly of stable structures that are helix and sheet structures. In the presence of E. coli membrane mimicking vesicles, the contents of unordered structures of combi-2 are converted to 100% 310-helix structures at high peptide concentration and 100% β-sheets at low peptide concentration. These data show that Pep-1 and combi-2 have a similar effect on E. coli 13

ACS Paragon Plus Environment

Page 15 of 32

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

Langmuir

membrane mimicking vesicles at low concentration. The mechanism of interaction between E. coli membrane mimicking vesicles and both peptides involves a conformation change, and a strengthening of H bonds as evidenced by the changes in wavenumbers of NH groups. In our experimental conditions, when a high or low amount of peptide is added to E. coli membrane, the changes in the corresponding FTIR spectra are small, which prompts us to say that both peptides interact with E. coli membrane mimicking vesicles in a nondestructive way. In peptidelipid interactions, the first target of cationic peptides is the negative regions of the membrane that are carbonyl and phosphate groups. These two regions are not significantly affected in our case. Thus we can say that the AMP Combi-2 acts as a CPP, in agreement with the literature11. Ultrastructural analysis. SEM technique was used to visualize the differences in conformation and depth levels of both peptides in E. coli membrane mimicking vesicles. The SEM images shown in panels A, B, C, D and E of figure 3 provide strong evidence that adding peptides to E. coli membrane mimicking vesicles induces superstructures. E. coli membrane mimicking vesicles present large amorphous aggregates with no ordered structures. No obvious fibrils or ribbons appear. SEM images of E. coli at low (panel B) and high (panel C) Pep-1 concentration show the presence of mostly dense and ordered fibril-like structures of a few micron length42,52–59. The surface of the layers is also smooth and homogeneous. SEM images of E. coli at low (panel D) and high (panel E) combi-2 concentration also show the presence of dense and ordered structures of a few micron length like those obtained with Pep-1. These results agree with the infrared results that show that complexes of Pep-1 or combi-2 with E. coli membrane mimicking vesicles are composed of helix and sheet structures. SEM images suggest that both peptides disrupt E. coli mimicking membranes by forming superstructures. Comparison of panels B, C, D, and E of figure 3 reveals that both 14

ACS Paragon Plus Environment

Langmuir

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 32

peptides have a similar effect on E. coli vesicles and these effects do not depend on peptide concentration. 3.3. Thermal analysis TGA and DSC were used to study the effects of adding the two peptides on the thermal stability of the E. coli vesicles. In general, from the TGA profiles, there are three main regimes of weight loss. The first one between room temperature and 200oC corresponds to the loss of weakly bound surface and interlayer water. The second one between 200oC and 450oC corresponds to the breaking of the bonds. This temperature range includes complex events such as decarboxylation, deamination, desulphyration, and dephosphorylation. The third endothermic event beyond 450oC consists of complete decomposition of the sample46. We keep in mind that complexes are most stable when a smaller loss of mass occurs over a given temperature range46. Figure 4 presents the TGA curves of E. coli and its complexes with Pep-1 and combi-2. At 50% weight loss, figure 4 reveals that Pep-1 – E. coli complexes at low and high peptide concentration lose 50% of their mass at a lower temperature than E. coli peptide-free and combi2 - E. coli complexes. Thus the thermal stability is in the order combi-2-E coli (high) > combi-2E. coli (low) > E. coli = Pep-1-E. coli (high) = Pep-1-E. coli (low). Samples of E. coli with and without Pep-1 have a similar mass loss at 50%, which indicates that there is no strong interaction between E. coli and Pep-1. TGA data demonstrate that CPPs interact with membrane mimicking vesicles in a nondestructive way1–4. Figure 5 presents the DSC profiles of E. coli and its complexes with Pep-1 and combi-2. To verify whether the peptides could discriminate between the components of the ternary mixture of phospholipids mimicking E. coli membrane mimicking vesicles, we compared the 250-400oC regions where the lipids appear. Figure 5 presents the DSC profile of E. coli mimicking vesicles, 15

ACS Paragon Plus Environment

Page 17 of 32

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

Langmuir

showing a broad and asymmetric peak with an enthalpy change of about 909 J/g corresponding to the breaking of the H and intermolecular bonds of the lipid mixture. The asymmetric property of the peak may be related to the non-ideal behavior of the ternary lipid mixture (CL:DPPG:DPPE). The low and high temperature peaks have almost the same amplitude. This peak is differently and dramatically affected by the presence of Pep-1 and combi-2. The results of low and high peptide concentrations are similar. In the presence of Pep-1, the low temperature peak decreases in amplitude while the high temperature peak becomes higher and sharper, and completely dissymmetric. These data suggest that Pep-1 discriminates between the lipid components; the low temperature peak corresponds to the peptide-poor domain and the high temperature peak to the peptide-rich domain, a phenomenon already seen in the literature49. Also, the total enthalpy has increased from 889 J/g to 1812 J/g, suggesting a stabilizing effect of the model membranes by Pep-1 at high peptide concentration. In contrast, the total enthalpy has decreased from 889 J/g to 288 J/g, suggesting a destabilizing effect of the model membranes by Pep-1 at low peptide concentration. For combi-2, the total enthalpy has increased from 889 J/g to 1245 J/g and 1134 J/g suggesting a stabilizing effect of the model membranes at high and low peptide concentration, respectively. A striking feature about combi-2 is that the peak of E. coli is split into two parts. This may suggest that combi-2 interacts exclusively with the negatively charged lipids that are CL and DPPG, ignoring the neutral portion of DPPE. Overall, our studies agree with that of Vogel et al.7 suggesting that the bactericidal action of the peptide combi-2 may involve translocation across the membrane, and there is no substantial leakage from vesicles. Pep-1 and combi-2 are both CPPs and AMPs at low concentration. With respect to E. coli, the increased stability of combi-2-E. coli complexes may be due to the small size of combi-2 with respect to Pep-1, leading to a finer balance between π-π stacking of 16

ACS Paragon Plus Environment

Langmuir

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 32

tryptophan residues, electrostatic effects due to arginine and steric effects. From the point of view of mechanism of interaction, this study suggests that the negative region of membrane is the target of the antimicrobial peptide combi-2 through the presence of arginine residues. This preferential membrane interaction of combi-2 may provide an insight into the studies of peptide and bacterial membrane interactions. The peaks are even sharper with combi-2 than with Pep-1, implying more ordered structures with combi-2 as seen in SEM images. The stronger interaction of combi-2 compared to Pep-1 was expected as combi-2 has two tryptophan and two arginine residues while Pep-1 has five lysine residues. Arginine residue causes stronger bonds than lysine residue43,54. Thus, the mechanisms of interaction of peptides are different, resulting to a similar conformation within the E. coli mimicking membrane. Using fluorescence on liquid samples, we observed a blue shift and a decrease in intensity. This allows us to infer that Pep-1 lies inside the hydrophobic region and combi-2 in the interfacial region. On the other hand, FTIR results show that the phosphate group (negative) peaks are not affected by the presence of combi-2 and Pep-1 which have a positive arginine residue. This implies both peptides are not in the same region as the phosphate group. Since we are getting the same conclusion from both liquid and solid samples, we assume that the structures of the liquid samples are close to those of the solid samples. The present report is a study of vesicles as mimetic agents. The results herein could differ with interaction of peptides with real E. coli membranes due to multitude of components missing in the mimicking vesicles such as cholesterol and sphingolipids. Natural membranes are very complex, and the use of simple models such as those included in this study provides detailed information of biological membranes. The methods used here give many details on the targeting interaction between peptides and lipids. 17

ACS Paragon Plus Environment

Page 19 of 32

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

Langmuir

CONCLUSIONS We have expanded the application of fluorescence, FTIR, TGA, DSC and SEM techniques on the self-assembly behavior of Pep-1 and combi-2 and the resultant superstructures in the presence of model membranes. This study provides a better understanding of the molecular interactions between Pep-1 and combi-2 and model membranes. Vibrational data revealed a multiplicity of secondary structures corresponding to a multiplicity of peaks in TGA/DSC profiles related to various morphologies shown by SEM. We have established that both peptides show a preferential interaction with one of the anionic lipids and are able to disrupt the E. coli membrane. On one hand, the ability of Pep-1 and combi-2 to form superstructures with E. coli model membranes seems to be a requirement for their activity. On the other hand, the ability of both peptides to form a fibrillar structure in solid state is similar to that of amyloid fibrils. Thus, the fibrillar morphology of these peptides may be used as a model system to study the fibrillation process of many neurotoxic diseases causing amyloid fibrils. The presence of a helical conformation in two helical tracts may also offer a new direction in the studies of fibrils in neurodegenerative diseases. Finally, this approach may be useful in creating new materials in nanotechnology by self-assembly. Also, the methods used here are likely to be applicable to a broad range of lipid mixtures. Future studies may include an in-depth study of peptide-lipid interactions focusing on the role of the size of the headgroups of the lipids in the mixture.

Supporting Information for Publication Supplemental Figure S1: Fluorescence emission spectra of Pep-1 in the presence of E coli lipid mixture as the lipid/ peptide ratio is increased from 0 to 40. 18

ACS Paragon Plus Environment

Langmuir

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

Supplemental Figure S2: Fluorescence emission spectra of Combi-2 in the presence of E coli lipid mixture as the lipid/ peptide ratio is increased from 0 to 40.

19

ACS Paragon Plus Environment

Page 20 of 32

Page 21 of 32

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

Langmuir

FIGURES

Figure 1. Blueshifts in tryptophan emission maxima of Pep-1 and combi-2 in Escherichia Coli membrane mimetic vesicles.

20

ACS Paragon Plus Environment

Langmuir

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 2. FTIR spectra of lyophilized samples of Pep-1 and combi-2 in the presence and absence of Escherichia Coli membrane mimetic vesicles in the carbonyl and amide I and II region.

21

ACS Paragon Plus Environment

Page 22 of 32

Page 23 of 32

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

Langmuir

Figure 3. SEM images of lyophilized samples of E. coli (panel A), of lyophilized samples of (E. coli-Pep-1 complexes at low and high peptide concentration (panels B,C), and of lyophilized samples of E. coli-combi-2 complexes at low and high peptide concentration (panels D,E) .

22

ACS Paragon Plus Environment

Langmuir

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 4. TGA curves for lyophilized samples of Escherichia Coli membrane mimetic vesicles and its complexes with Pep-1 and combi-2.

23

ACS Paragon Plus Environment

Page 24 of 32

Page 25 of 32

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

Langmuir

Figure 5. DSC thermograms for lyophilized samples of Escherichia Coli membrane mimetic vesicles and its complexes with Pep-1 and combi-2.

24

ACS Paragon Plus Environment

Langmuir

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 32

TABLES Table 1. Wavenumber characteristics of the main functional groups of Pep-1, combi-2 and their complexes with E. coli model membranes

Assignment

Approximate E. coli Wavenumber

Pep-1

Pep-1Pep-1CombiE. coli E. coli 2 [1:1] [1:40]

Combi- Combi2-E. coli 2-E. coli [1:1] [1:40] 3346

3350

Range (cm-1) N-H stretch

3100-3400

3374

3276

3295

3363

C-H 2900-2950 antisymmetric stretch

2917

2936

2918

2918

2918

2918

C=O stretch

1700-1750

1741

1740

1738

1739

1738

PO2asymmetric stretch

1200-1240

1223

1222

1222

1222

1222

PO2symmetric stretch

1050-1100

1079

1079

1078

1078

1078

25

ACS Paragon Plus Environment

32763190

Page 27 of 32

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

Langmuir

AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors declare no competing financial interest. ACKNOWLEDGMENT Nsoki Phambu wants to express his deepest gratitude to Tennessee State University for the support. We thank Mr. Richard Graves (Xavier-RCMI/Drug Delivery Core- NIH Grant #8G12MD007595) for technical assistance with Scanning Electron Microscope. This material is based upon work supported by the National Science Foundation HBCU-UP and Research Initiation Awards under Grants No. 1036147 and 1411209. ABBREVIATIONS SEM, scanning electron microscopy; FTIR, Fourier transform infrared: TGA, thermogravimetric analysis; DSC, differential scanning calorimetry; Dimyristoylphosphotidylethanolamine (DPPE); Dimyristoylglycerophosphorylglycerol (DPPG); Cardiolipin (CL)

26

ACS Paragon Plus Environment

Langmuir

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 32

REFERENCES (1)

(2)

(3) (4)

(5) (6)

(7) (8)

(9)

(10) (11)

(12)

(13)

(14) (15)

Deshayes, S.; Heitz, A.; Morris, M. C.; Charnet, P.; Divita, G.; Heitz, F. Insight into the Mechanism of Internalization of the Cell-Penetrating Carrier Peptide Pep-1 through Conformational Analysis. Biochemistry (Mosc.) 2004, 17 (43), 1449–57. Alves, I. D.; Correia, I.; Jiao, C. Y.; Sachon, E; Sagan, S.; Lavielle, S.; Tollin, G.; Chassaing, G. The Interaction of Cell-Penetrating Peptides with Lipid Model Systems and Subsequent Lipid Reorganization: Thermodynamic and Structural Characterization. J Pept Sci 2009, 15, 200–209. Ding, B.; Chen, Z. Molecular Interactions between Cell Penetrating Peptide Pep-1 and Model Cell Membranes. J Phys Chem B 2012, 116 (8), 2545–2552. Jobin, M.-L.; Alves, I. D. On the Importance of Electrostatic Interactions between Cell Penetrating Peptides and Membranes: A Pathway toward Tumor Cell Selectivity? Biochimie 2014, 107, Part A, 154–159. Schmidt, N.; Mishra, A.; Lai, G. H.; Wong, G. C. L. Review: Arginine-Rich CellPenetrating Peptides. FEBS Lett. 2010, 584, 1806–1813. Nowotarska, S. W.; Nowotarski, K. J.; Friedman, M.; Situ, C. Effect of Structure on the Interactions between Five Natural Antimicrobial Compounds and Phospholipids of Bacterial Cell Membrane on Model Monolayers. Molecules 2014, 19 (6), 7497–7515. Vogel, H. J. Interactions of the Antimicrobial Peptide Ac-FRWWHR-NH2 with Model Membrane Systems and Bacterial Cells. J Pept. Res 2005, 65, 491–501. Jing, W.; Hunter, H. N.; Hagel, J.; Vogel, H. J. The Structure of the Antimicrobial Peptide Ac-RRWWRF-NH2 Bound to Micelles and Its Interactions with Phospholipid Bilayers. J Pept Res 2003, 61 (5), 219–29. Liu, Z.; Young, A. W.; Hu, P.; Rice, A. J.; Zhou, C.; Zhang, Y.; Kallenbach, N. Length Effects in Antimicrobial Peptides of the (RW)n Series. Antimicrob Agents Chemother 2007, 51, 597–603. Chan, D. I.; Prenner, E. J.; Vogel, H. J. Tryptohan- and Arginine-Rich Antimicrobial Peptides: Structure and Mechanisms of Action. Biochim Biophys Acta 2006, 1758–1184. Joanne, P.; Galanth, C.; Goasdoué, N.; Nicolas, P.; Sagan, S.; Lavielle, S.; Chassaing, G.; El Amri, C.; Alves, I. D. Lipid Reorganization Induced by Membrane-Active Peptides Probed Using Differential Scanning Calorimetry. Biochim. Biophys. Acta 2009, 1788 (9), 1772–1781. Bi, X.; Wang, C.; Dong, W.; Zhu, W.; Shang, D. Antimicrobial Properties and Interaction of Two Trp-Substituted Cationic Antimicrobial Peptides with a Lipid Bilayer. J. Antibiot. (Tokyo) 2014, 67 (5), 361–368. Morein, S.; Koeppe, R. E.; lindblom, G.; de Kruijff, B.; Killian, J. A. The Effect of Peptide/Lipid Hydrophobic Mismatch on the Phase Behavior of Model Membranes Mimicking the Lipid Composition in Eschericia Coli Membranes. Biophys. J. 2000, 78, 2475–2485. Meister, A.; Finger, S.; Hause, G.; Blume, A. Morphological changes of bacterial model membrane vesicles. Eur. J. Life Sci. Technol. 2014, 116, 1228–1233. Borle, F.; Seelig, J. Structure of Escherichia Coli Membranes. Deuterium Magnetic Resonance Studies of the Phosphoglycerol Head Group in Intact Cells and Model Membranes. Biochemistry (Mosc.) 1983, 22, 5536–5544. 27

ACS Paragon Plus Environment

Page 29 of 32

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

Langmuir

(16) Arouri, A.; Dathe, M.; Blume, A. Peptide Induced Demixing in PG/PE Lipid Mixtures: A Mechanism for the Specificity of Antimicrobial Peptides towards Bacterial Membranes? Biochim. Biophys. Acta 2009, 1788, 650–659. (17) Matos, P. M.; Franquelim, H. G.; Castanho, M. A. R. B.; Santos, N. C. Quantitative Assessment of Peptide–lipid Interactions. Ubiquitous Fluorescence Methodologies. Biochim. Biophys. Acta 2010, 1798, 1999–2012. (18) Surewicz, W. K.; Epand, R.M. Role of Peptide Structure in Lipid-Peptide Interactions: A Fluorescence Study of the Binding of Pentagastrin-Related Pentapeptides to Phospholipid Vesicles. Biochemistry (Mosc.) 1984, 23 (25), 6072–6077. (19) Prenner, E. J.; Lewis, R. N. A. H.; McElhaney, R. N. The Interaction of the Antimicrobial Peptide Gramicidin S with Lipid Bilayer Model and Biological Membranes. Biochim. Biophys. Acta BBA - Biomembr. 1999, 1462 (1–2), 201–221. (20) Loura, L. M. S.; de Almeida, R. F. M.; Coutinho, A.; Prieto, M. Interaction of Peptides with Binary Phospholipid Membranes: Application of Fluorescence Methodologies. Chem. Phys. Lipids 2003, 122, 77–96. (21) Capdevila, F. R. D. A.; Rouco, S. O.; Tomasina, F.; Tortora, V.; Demicheli, V.; Radi, R.; Murgida, D. H. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin. Biochemistry (Mosc.) 2015, 54, 7491–7504. (22) Dreissig, I.; Machill, S.; Salzer, R.; Krafft, C. Quantification of Brain Lipids by FTIR Spectroscopy and Partial Least Squares Regression. Spectrochim. Acta Part 71 2009, 2069–2075. (23) Schultz, Z. D.; Levin, I. W. Vibrational Spectroscopy of Biomembranes. Annu. Rev Anal Chem 2011, 4, 343–366. (24) Lewis, R. N. A. H.; Zhang, Y.-P.; McElhaney, R. N. Calorimetric and Spectroscopic Studies of the Phase Behavior and Organization of Lipid Bilayer Model Membranes Composed of Binary Mixtures of Dimyristoylphosphatidylcholine and Dimyristoylphosphatidylglycerol. Biochim. Biophys. Acta BBA - Biomembr. 2005, 1668 (2), 203–214. (25) Benesch, M. G. K.; Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. A Calorimetric and Spectroscopic Comparison of the Effects of Lathosterol and Cholesterol on the Thermotropic Phase Behavior and Organization of Dipalmitoylphosphatidylcholine Bilayer Membranes. Biochemistry (Mosc.) 2011, 50 (46), 9982–9997. (26) Ionov, M.; Klajnert, B.; Gardikis, K.; Hatziantoniou, S.; Palecz, B.; Salakhutdinov, B.; Cladera, J.; Zamaraeva, M.; Demetzos, C.; Bryszewska, M. Effect of Amyloid Beta Peptides Aβ1–28 and Aβ25–40 on Model Lipid Membranes. J. Therm. Anal. Calorim. 2009, 99 (3), 741–747. (27) Ivanova, V. P.; Makarov, I. M.; Schaffer, T. E.; Heimburg, T. Analyzing Heat Capacity Profiles of Peptide-Containing Membranes: Cluster Formation of Gramicidin. In A. Biophysical Journal Volume 84; 2003; pp 2427–2439. (28) Oliva, R.; Del Vecchio, P.; Stellato, M. I.; D’Ursi, A. M.; D’Errico, G.; Paduano, L.; Petraccone, L. A Thermodynamic Signature of Lipid Segregation in Biomembranes Induced by a Short Peptide Derived from Glycoprotein gp36 of Feline Immunodeficiency Virus. Biochim. Biophys. Acta 2015, 1848, 510–517. (29) Prenner, E. J.; Lewis, R. N.; Kondejewski, L. H.; Hodges, R. S.; McElhaney, R. N. Differential Scanning Calorimetric Study of the Effect of the Antimicrobial Peptide 28

ACS Paragon Plus Environment

Langmuir

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

(30)

(31) (32)

(33)

(34)

(35)

(36) (37)

(38)

(39)

(40) (41) (42)

(43)

Page 30 of 32

Gramicidin S on the Thermotropic Phase Behavior of Phosphatidylcholine, Phosphatidylethanolamine and Phosphatidylglycerol Lipid Bilayer Membranes. Biochim Biophys Acta 1999, 4 (1417), 211–23. Epand, R. F.; Schmitt, M. A.; Gellman, S. H.; Epand, R. M. Role of Membrane Lipids in the Mechanism of Bacterial Species Selective Toxicity by Two α/β-Antimicrobial Peptides. Biochim. Biophys. Acta 2006, 1758, 1343–1350. Taylor, K. M. G.; Morris, R. M. Thermal Analysis of Phase Transition Behavior in Liposomes. Thermochim. Acta 1995, 248, 289–301. Dandurand, J.; Samouillan, V.; Lacoste-Ferre, M. H.; Lacabanne, C.; B.Bochicchio, A. P. Conformational and Thermal Characterization of a Synthetic Peptidic Fragment Inspired from Human Tropoelastin: Signature of the Amyloid Fibers. Pathol. Biol. 2014, 62, 100– 107. Sunooj, K. V.; George, J.; Kumar, V. A. S.; and A. S. Bawa, K. R. Thermal Degradation and Decomposition Kinetics of Freeze Dried Cow and Camel Milk as Well as Their Constituents. J. Food Sci. Eng. 2011, 1, 77–84. Adler-Abramovich, L.; Reches, M.; Sedman, V. L.; Allen, S.; Tendler, S. J. B.; Gazit, E. Thermal and Chemical Stability of Diphenylalanine Peptide Nanotubes:  Implications for Nanotechnological Applications. Langmuir 2006, 22 (3), 1313–1320. Surmacz-Chwedoruk, W.; Malka, I.; Bozycki, L.; Nieznanska, H.; Dzwolak,W. On the Heat Stability of Amyloid-Based Biological Activity: Insights from Thermal Degradation of Insulin Fibrils. PLOS ONE 9 2014, 1, 286320. Lim, J. J.; Shamos, M. H. Evaluation of Kinetic Parameters of Thermal Decomposition of Native Collagen by Thermogravimetric Analysis. Biopolymers 1974, 13 (9), 1791–1807. Nascimento, J. M.; Oliveira, M. D. L.; Franco, O. L.; Migliolo, L.; de Melo, C. P.; Andrade, C. A. S. Elucidation of Mechanisms of Interaction of a Multifunctional Peptide Pa-MAP with Lipid Membranes. Biochim. Biophys. Acta BBA - Biomembr. 2014, 1838 (11), 2899–2909. Leung, B. O.; Hitchcock, A. P.; Won, A.; Ianoul, A.; Scholl, A. Imaging Interactions of Cationic Antimicrobial Peptides with Model Lipid Monolayers Using X-Ray Spectromicroscopy. Eur. Biophys. J. EBJ 2011, 40 (6), 805–810. Park, S.-C.; Kim, M.-H.; Hossain, M. A.; Shin, S. Y.; Kim, Y.; Stella, L.; Wade, J. D.; Park, Y.; Hahm, K.-S. Amphipathic α-Helical Peptide, HP (2–20), and Its Analogues Derived from Helicobacter Pylori: Pore Formation Mechanism in Various Lipid Compositions. Biochim. Biophys. Acta BBA - Biomembr. 2008, 1778 (1), 229–241. Hung, W.C.; Lee, M.T. The Interaction of Melittin with E. Coli Membrane: The Role of Cardiolipin. Chin. J. Phys. 2006, 44 (2), 137–149. Jackson, M.; Mantsch, H. H. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Crit Rev Biochem Mol Biol 1995 (30), 95–120. Frias, M.; Benesch, M. G. K.; Lewis, R. N. A. H.; McElhaney, R. N. On the Miscibility of Cardiolipin with 1,2-Diacyl Phosphoglycerides: Binary Mixtures of Dimyristoylphosphatidylethanolamine and Tetramyristoylcardiolipin. Biochim. Biophys. Acta BBA - Biomembr. 2011, 1808 (3), 774–783. Liberato, M. S.; KogikoskiJr., S.; Silva, E. R.; Coutinho-Neto, M. D.; Scott, L. P. B.; Silva, R. H.; OliveiraJr., V. X.; Ando, R. A.; Alves, W. A. Self-Assembly of Arg–Phe Nanostructures via the Solid–Vapor Phase Method. J Phys Chem B 2013, 117 (3), 733– 740. 29

ACS Paragon Plus Environment

Page 31 of 32

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

Langmuir

(44) Tian, F.; Sane, S.; Rytting, J. H. Calorimetric Investigation of Protein/Amino Acid Interactions in the Solid State. Int. J. Pharm. 2006, 310, 175–186. (45) Rodate, F.; Marrosu, G. Thermal Analysis of Some α-Amino Acids Using Simultaneous TG-DSC Apparatus. The Use of Dynamic Thermogravimetry to Study the Chemical Kinetics of Solid State Decomposition. Thermochim. Acta 1990, 171 (24), 15–29. (46) Gorinstein, S. A Thermogravimetric Study of the Stability under Heat of Iron-Protein Complexes. J Agr Food Chem 1975, 23, 1–45. (47) Li, G.; Huang, Y.; Feng, Q.; Chen, Y. Tryptophan as a Probe to Study the Anticancer Mechanism of Action and Specificity of α-Helical Anticancer Peptides. Molecules 2014, 19, 12224–12241. (48) Contreras, L. M.; de Almeida, R. F.; Villalaín, J.; Fedorov, A.; Prieto, M. Interaction of Alpha-Melanocyte Stimulating Hormone with Binary Phospholipid Membranes: Structural Changes and Relevance of Phase Behavior. Biophys. J. 2001, 80 (5), 2273– 2283. (49) Misiewicz, J.; Afonin, S.; Ulrich, A. S. Control and role of pH in peptide-lipid interactions in oriented membrane samples. Biochim. Biophys. Acta 2015, 1848, 833–841. (50) Zhou, S.; Peng, H.; Yu, X.; Zheng, X.; Cui, W.; Zhang, Z.; Li, X.; Wang, J.; Weng, J.; Jia, W.; Li, F.. Preparation and Characterization of a Novel Electrospun Spider Silk Fibroin/Poly(D,L-Lactide) Composite Fiber. J Phys Chem B 2008, 112 (36), 11209– 11216. (51) Du, Z.; Guan, S. J.; Yao, S. J.; Zhu, Z. Q. Supercritical Fluid Assisted Atomization Introduced by an Enhanced Mixer for Micronization of Lysozyme: Particle Morphology, Size and Protein Stability. Int. J. Pharm. 2011, 421, 258–268. (52) Yamaoki, Y.; Imamura, H.; Fulara, A.; Wójcik, S.; Bożycki, Ł.; Kato, M.; Keiderling, T. A.; Dzwolak, D. An FTIR Study on Packing Defects in Mixed β-Aggregates of Poly(LGlutamic Acid) and Poly(D-Glutamic Acid): A High-Pressure Rescue from a Kinetic Trap. J Phys Chem B 2012, 116 (17), 5172–5178. (53) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Studies of Peptides Forming 310and α-Helixes and β-Bend Ribbon Structures in Organic Solution and in Model Biomembranes by Fourier Transform Infrared Spectroscopy. Biochemistry 1991, 30 (26), 6541–6548. (54) Dutta, A.; Kar, S.; Fröhlich, R.; Koley, P.; Pramanik, A. Studies of the β-Sheet Mediated Self-Assembly of Designed Synthetic Peptides of General Formula PhCO-Gly-XxOCH2Ph and the Possible Role of Aromatic π-π Interactions in the Self-Assembly. ARKIVOC 2009, 247–259. (55) Aronsson, C.; Selegård, R.; Aili, D. Zinc-Triggered Hierarchical Self-Assembly of Fibrous Helix–Loop–Helix Peptide Superstructures for Controlled Encapsulation and Release. Macromolecules 2016, 49 (18), 6997–7003. (56) Choi, S.; Swanson, J. M. Interaction of Cytochrome c with Cardiolipin: An Infrared Spectroscopic Study. Biophys. Chem. 1995, 54, 271–278. (57) Fulara, A.; Lakhani, A.; Wojcik, S.; Nieznanska, H.; Keiderling, T. A.; Dzwolak, W. Spiral Superstructures of Amyloid-Like Fibrils of Polyglutamic Acid: An Infrared Absorption and Vibrational Circular Dichroism Study. J Phys Chem B 2011, 115, 11010– 11016.

30

ACS Paragon Plus Environment

Langmuir

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

(58) Kar, S.; Wu, K. W.; Hsu, I. J.; Lee, C. R.; Tai, Y. Study of the Nano-Morphological Versatility by Self-Assembly of a Peptide Mimetic Molecule in Response to Physical and Chemical Stimuli. Chem Commun 2014, 50, 2638–2641. (59) Han, S.; Lee, M.; Lim, Y. Cell-Penetrating Cross-β Peptide Assemblies with Controlled Biodegradable Properties. Biomacromolecules 2017, 18 (1), 27–35.

31

ACS Paragon Plus Environment

Page 32 of 32