Deprotonation of a Single Amino Acid Residue Induces Significant

Subscriber access provided by CARLETON UNIVERSITY

Article

Deprotonation of a Single Amino Acid Residue Induces Significant Stability in an #-Helical Heteropeptide Gouri S. Jas, and Krzysztof Kuczera J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07418 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 15, 2018

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 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 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.

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 50 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

The Journal of Physical Chemistry

Figure 1.

ACS Paragon Plus Environment

The Journal of Physical Chemistry 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.


Page 2 of 50

Page 3 of 50 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.



Figure 4.


Page 4 of 50

Page 5 of 50 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.

Figure 6.



Figure 7.


Page 6 of 50

Page 7 of 50 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.



Figure 9.


Page 8 of 50

Page 9 of 50 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 10.



Figure 11.


Page 10 of 50

Page 11 of 50 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 12.



Figure 13.


Page 12 of 50

Page 13 of 50 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 14.



Deprotonation of a Single Amino acid Residue Induces Significant Stability in an -Helical Heteropeptide

Gouri S. Jas*1 and Krzysztof Kuczera2,3

*Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047 2

Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66047

3

Department of Chemistry, The University of Kansas, Lawrence, KS 66045

1

Corresponding Author:

Gouri S. Jas Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047 [email protected]


Page 14 of 50

Page 15 of 50 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. Stability of secondary structural elements is an integral component of a structurally stable protein. Presence of protons in the residue sequence and their immediate environment play a significant role in conformational stability. In this study, we show that removing a proton from a single amino acid residue significantly increases the stability of an alpha helical heteropeptide in comparison with the unprotonated form. Far-UV circular dichroism spectroscopy, fluorescence spectroscopy, fluorescence energy transfer measurements and over ten microseconds of all-atom molecular dynamics simulations are used to provide an atomically detailed characterization of this event. There is a single histidine residue in the studied alpha-helical peptide sequence towards the N-terminal that interacts with a tryptophan located four residues away and quenches the fluorescence when protonated. Removing a proton from this histidine residue dequenches the tryptophan fluorescence and contributes to a significant increase in the helix stability. Atomically detailed analysis of individual residue conformations shows that the protonated histidine tends to be in closer proximity to the tryptophan, which correlates with higher helix content in the N and C termini and lower helix content in the central region of the peptide. In the presence of a neutral histidine, when tryptophan fluorescence is no longer quenched and histidine moves further away from tryptophan, the helix content remains mostly unchanged in the N-and-C termini and significantly increases in the central region. Our results strongly suggest that interactions of the tryptophan with a protonated histidine downregulate the helix population in the central segment of the helical structure compared to a neutral histidine residue. Upregulation of helix population of the central segment of this -helical heteropeptide in the presence of a neutral histidine residue, significantly increases the peptide structural stability.



Introduction. The most common secondary structural elements in a stable biologically functional protein are helices1-3. A favorable backbone dihedral angle (-) pattern following a parallel hydrogen bond formation are important driving force in the formation of helical structures. Based on the hydrogen bond formation pattern the helical structures are subclassified as 3-10, , and  In a 3-10,   helical structure the hydrogen bond formation takes place based on the interaction between the first and the third, the first and the fourth, and the first and the fifth residue along the residual chain sequences, respectively3. Our current study focuses on a system with the interaction between first and the fifth residue in a chain length of twenty-one residues4-7. The N- and C-termini of this chain are capped with an acetyl and an amide group, respectively. Amino acid residues vary in their propensity to from stable helix with alanine found to have highest propensity to form a stable helix in solution4, 8-9. In our studied system, the first residue is a tryptophan located four residues away from a histidine interspaced with three alanine residues. Tryptophan is used as a fluorescence probe and the titratable histidine can quench and dequench tryptophan fluorescence based on the environmental pH10-16. At a lower pH, like pH 4.8, this histidine is protonated and quenches tryptophan fluorescence providing information about specific structural conformation of the system. At higher pH, like pH 7.2, this His residue exists in a neutral form and dequenches tryptophan fluorescence. It would be interesting to understand how this tryptophan-histidine interaction plays a role in the overall stability of the helical system. As the protonated histidine residue moves closer to the indole ring of tryptophan with its delocalized electron cloud and quenches the fluorescence, one can speculate that this strong physical interaction may add additional stress to the overall system and may trigger a destabilization of the overall structure. If this approach is valid then one would consider deprotonation and make the histidine neutral to release the stress of this interaction with tryptophan and observe the influence on the overall structural stability. It is this philosophy that drove us to the current combined experimental and computational study. In order to address these concerns, we employed far-UV circular dichroism spectroscopy to monitor change in overall structural stability of a helix in its protonated and neutral form. Steadystate temperature dependent fluorescence spectroscopy is used to monitor the quenching effect on the tryptophan of the two different ionic form of this helical system. We have added a dansylated lysine group at the C-terminal end of this helical system to apply fluorescence energy transfer to monitor changes in the end-to-end distance as a function of temperature and thus observe the


Page 16 of 50

Page 17 of 50 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


pattern of the unfolding pathway of this system under two different ionization states. All-atom molecular dynamics simulation with two different forcefields are carried out on two different ionic forms of this -helical peptide system to obtain an atomically detailed insight into the mechanism that governing the principle of increasing stability on the studied system. Here we present a combinatorial picture with a set of detailed experimental measurements and comprehensive computational study in the two different ionic forms of a model 21-residue αhelical heteropeptide (WH21/WH21n). In our experimental component, we have measured farUV circular dichroism spectroscopy to generate thermal denaturation curves of two different ionic forms, with a neutral and a protonated histidine, to monitor the change in stability. It is observed that the helix is stabilized upon removing a proton from histidine with a melting temperature increased by about sixteen degrees with respect to the protonated form. With tryptophan as a donor and a dansylated lysine as an acceptor, we employed fluorescence resonance energy transfer (FRET) as a function of temperature to observe the changes in end-to-end distance thus obtain a detailed picture about the pattern of the unfolding pathway. FRET results show distinct differences in the unfolding pathways of the two ionization states of this -helical peptide under two different ionic conditions, with an initial decrease in length followed by an increase in the protonated form, and a flat profile followed by increase in the neutral peptide. In the computational aspect of this study, we have carried microsecond length replica-exchange molecular dynamics simulations as well as all molecular simulation of this system with the neutral and protonated form of histidine in explicit solvent, providing an atomically detailed picture of the unfolding processes, the mechanism that governs the increase stability in the neutral form of histidine, and the changes in the kinetics in the two ionic forms of this -helical system. In both ionization forms of this αhelical peptide, we found a large heterogeneous population of intermediates, with unfolding starting at the termini and progressing through a stable helical region in the center of the peptide. The intermediates are a mixture and helix-turn-helix motifs (“broken helices”) and single helices with frayed ends. In the neutral form, the population of the helical state is significantly increased. A detailed microscopic picture emerged from over ten microseconds of all atom molecular dynamic in the neutral and protonated form of this system. In the protonated form, the tryptophanhistidine distance (Trp-His) is found to be much shorter, tryptophan stays closer to histidine as a function of time. In the neutral form the Trp-His distance is found to be much larger. Residue by



residue dihedral analysis showed a varying distribution of helix population across the helical length. In the N and C termini segmental area there are no significant changes in the helix population in these two ionic forms. However, a significant increase in helix population is observed in the mid-segment of the of the helix, spreading from residue six through residue eighteen in the neutral form. Hydrogen bond analysis presents a similar picture, with no significant changes the N and C termini area of this system in two ionic forms and a significant increase in hydrogen bond population residues ranging from six through eighteen in the neutral form. Kinetic analysis of the protonated form of this system produced a relaxation time of about 330ns at 300 K which is found to be in very good agreement with the experimentally measures relaxation time of 300 ns at 300K. Kinetic analysis of the neutral form of this system produced a significantly increased relaxation time, about a factor three greater at 300 K in comparison with the protonated form. This combined study of experiment and molecular dynamics simulation presented very consistent pictures, uncovering large changes in observed peptide properties with pH and providing a detailed microscopic mechanism for understanding the governing principle of the experimental observables. Methods. Materials. The 21-residue helical heteropeptide Ac-WAAAH-(AAARA)3A-NH2 (WH21)4-7 and its dansylated form, Ac-WAAAH-(AAARA)3AK-(dansyl)-NH2 (dan-WH21) were obtained from GenScript Corporation (Piscataway, NJ, USA) with a >95% purity. Experimental measurements on WH21/WH21n were carried by dissolving in 20 mM acetate buffer of pH 4.8 and 20 mM phosphate buffer of pH 7.2. Measured concentrations of each samples were determined with the absorbance of tryptophan at 280 nm and a molar extinction coefficient of 5690 M-1cm-1. Experiments. Circular Dichroism. Far UV circular dichroism spectroscopy as a function of temperature of the non-dansylated and dansylated form were measured on a JASCO 815 spectropolarimeter (Tokyo, Japan) in a cylindrical cell with a pathlength of 0.5 mm and concentrations of ~220 µM. Scans between 266-360 K were recorded in a 10degree increments. Denaturation curves were measured at 222 nm for all WH21/WH21n. Thermodynamic parameters and fraction helix as a function of temperature were obtained from the melting curve with a two-state equilibrium model EXAM.17 The two-state fit obtained temperature-independent parameter of the transition enthalpy ΔH° and


Page 18 of 50

Page 19 of 50 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


entropy ΔS°, and the mid-point temperature Tm = ΔH°/ΔS°. The Gibbs free energy was calculated as ΔG° = ΔH° − TΔS°, and the equilibrium constant K was found through ΔG° = −RT ln(K). With a conservative estimate, the errors in these thermodynamic parameters are estimated to be ~5%, similar to previous analyses.4-7 Singular value decomposition (SVD) was applied to the 170− 260 nm CD scans to resolve overlapping spectral components. Fluorescence Spectroscopy. Fluorolog (Edison, NJ, USA) was used in the measurements of tryptophan fluorescence and FRET in a tryptophan dynsyl system. Excitation wavelength for tryptophan was 280 nm and the emission spectra were measured between 285 to 525 nm for the non-dansylated system and between 285 to 800 nm for the dansylated system. Temperature dependent spectra were recorded at a 5 degree increments between 268-363 K with a slit width 2 nm and an integration time of 1.0 second at a sample concentration of ~15M.

Donor-acceptor Distance.

Detail procedure was published elsewhere18. Briefly, The Förster

equation relating to energy transfer efficiency between the donor and acceptor (E) with distance (r): r = Ro[(1/E) – 1]1/6

(1)

Where Ro is the Förster radius at which the transfer efficiency is 50% and specific for a particular donor-acceptor pair. Experimentally determined Förster radius 21 Å for the Trp and dansyl pair was used. The Förster radius is found experimentally through: Ro = (Jκ2Qon-4)1/6x9.7x103

(2)

Where J is the spectral overlap integral between donor and acceptor; κ2 is the donor and acceptor dipole orientation, n is the refractive index of the medium between the donor and acceptor, and Qo is the quantum yield of the energy donor in the absence of acceptor.10-12 The energy transfer efficiency is derived from: E = 1-FDA/FD

(3)

Where FDA is the donor fluorescence intensity in the presence of the acceptor and FD is the donor fluorescence intensity in the absence of acceptor.5,19-21

FDA and FD were obtained from the

integrated fluorescence intensity between 310-440 nm as the indole emission maximum is around 363 nm.19



Computational methods. The simulated peptides were Ac-WA3H+-(AAARA)3-A-NH2 (WH21) and its form with a neutral histidine Ac-WA3H-(AAARA)3-A-NH2 (WH21n). For the simulations described here, extended conformations of the peptides were constructed with CHARMM22. The systems were then solvated with TIP3P water23 and ions in a cubic box of ca. 48 Å and briefly equilibrated with GROMACS24. MD trajectories of 12 μs length for WH21 and 13 μs length for WH21n were generated. From the same starting structures, replica-exchange MD (REMD) simulations were also performed, using 40 replicas spanning 300-500 K, with exchange attempts every 1 ps. The REMD trajectory lengths were 850 ns for WH21 and 1,000 ns for WH21n. MD and REMD simulations were carried out with GROMACS24 version 5 with the CHARMM22,25 36 force field (further denoted by C36). Additional computational details are described in the Supplementary Information. Hydrogen bonds were counted as formed when the O…N distance was below 3.6 Å and a residue was considered to be in the α-helical conformation when the values of (φ,ψ) were within a 20o radius of the ideal structure (-62o,-41o). Structure clustering was done with the gromos algorithm. Statistical error estimates were obtained by dividing data into ten consecutive blocks, and calculating the standard error of the mean at 95% confidence level. To analyze peptide kinetics time autocorrelation functions C1(t) = were calculated, for x being the number of α–helical hydrogen bonds and number of residues in the αhelical region of the Ramachandran map. These functions were fitted to two-exponential decays a*exp(-t/τ1)+(1-a)*exp(-t/τ2), and the longer time scale τ2 was used to estimate the global relaxation time of the peptide.

Results and Discussion. Experimental results. Far UV circular dichroism (CD) spectra of non-protonated and protonated WH21 with neutral (His0) and protonated (His+) histidine are presented in Figure 1. Figure 1A, shows the CD spectra of His0 as a function of temperature. Observed spectral features, with two minima at 222nm, 210nm and a maximum at 190nm, represent a classic alpha helical CD spectra with decrease helicity as a function of increasing temperature. Shown in Figure 1B is the CD spectral component analysis


Page 20 of 50

Page 21 of 50 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


with the application of singular value decomposition (SVD)7 on the complete set of temperature dependent CD spectra. Amplitudes of the first three components are presented with respect to wavelength. Presented in Figure 1C are the amplitude vectors of first three components corresponding to components shown in 1B, with respect to temperatures. A two-state fit to the amplitude vectors corresponding to the first component obtained a Tm of about 313K. Figure 1D shows the CD spectra of His+ as a function of temperature with two minima at 222nm, 210nm and a maximum at 190nm with decrease helicity as a function of increasing temperature. Shown in Figure 1E is the CD spectral component analysis with the application of SVD on the complete set of temperature dependent CD spectra. Amplitudes of the first three components are presented with respect to wavelength. Presented in Figure 1F are the amplitude vectors of first three components corresponding to components shown in 1E, with respect to temperatures. A two-state fit to the amplitude vectors corresponding to the first component obtained a Tm of about 297K. Thermal denaturation curve of non-protonated (neutral His0) and protonated (protonated His+) WH21 are presented in figure 2A. Molar ellipticity is measured as a function of increasing temperature at a fixed wavelength at 222nm. A two-state fit to these two melting curves with EXAM obtained a Tm for His0 and His+ are 313K and 297K, respectively. Fraction helix content as a function temperature is presented in Figure 2B. Fraction helix at 264K for His0 and His+ are 92.9 and 96.2, respectively. For His0 and His+ 50% helix contain are found to be present at 312.7K and 297.1K, respectively. Thermodynamic parameters from this analysis are presented in Table 1. Steady-state fluorescence spectra of as a function of temperature are measured in a protonated and neutral form of the studied -helical heteropeptide, shown in Figure 3. Presented in Figure 3A, are the temperature dependent fluorescence spectra of the protonated form, with decrease fluorescence intensity as a function of increasing temperature with emission maxima at 358nm. Shown in Figure 3B is the fluorescence spectral component analysis with the application of SVD26,27 on the complete set of temperature dependent fluorescence spectra. Amplitudes of the first three components are presented with respect to wavelength. Presented in Figure 3C are the amplitude vectors of first three components. The amplitude vectors of component one in a temperature range between 273K and 310K shows no significant decrease in the fluorescence intensity with increasing temperature suggesting a possible strong quenching of tryptophan fluorescence by the



protonated histidine. Above 310K, there is a steady drop in fluorescence intensity with increasing temperature. Presented in Figure 3D, are the temperature dependent fluorescence spectra of the neutral form, fluorescence intensity as a function of increasing temperature. Shown in Figure 3E is the components of temperature dependent fluorescence spectra after applying SVD on the complete set of temperature dependent fluorescence spectra. Amplitudes of the first three components are presented with respect to wavelength. Presented in Figure 3F are the amplitude vectors of first three components. The amplitude vectors of component one shows a steady sharp decrease throughout the spectral temperature range. In comparison with the first component amplitude vectors of the protonated form, the non-protonated form has a steady sharp decrease in the temperature range between 273K and 310K. Furthermore, in comparison with the shape of the first component amplitude vector of the protonated and non-protonated form with respect to temperature shows a significantly different feature, with protonated form showing a much shallower drop. This may suggest that a protonated form of histidine with much closer proximity to tryptophan quenches the fluorescence. In the non-protonated form this behavior is not present. In Figure 4, is shown the fluorescence energy transfer from donor tryptophan to the acceptor dansylated lysine in both protonated and non-protonated form of the helical heteropeptide. Figure 4A, shows the fluorescence energy transfer spectra of the neutral form of the peptide as a function of temperature. In Figure 4B, presented the SVD spectral features of first three components. Figure 4C, shows the amplitude vectors of first three components. Figure 4D, shows the fluorescence energy transfer spectra of the protonated form of the peptide as a function of temperature. In Figure 4E, presented the SVD spectral features of first three components. Figure 4E, shows the amplitude vectors of first three components. A comparison of the amplitude vectors of the first component in the protonated and non-protonated form shows similar behavior as observed in the nondansylated form. In order to confirm that the addition of a dansyl group in the c-terminal of helical peptide did not perturb the thermodynamic stability, we measured far UV CD of the protonated and nonprotonated form of the dansylated helical peptide. In Figure 5A, is presented the thermal denaturation curve of the protonated and neutral form of the dansylated peptide. A two-state fit was employed to obtain the thermodynamic parameters. Figure 5B, shows the fraction helix as a function of temperature of these two forms of dansylated helical peptide with the presence of a


Page 22 of 50

Page 23 of 50 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


50% helix content at about 300K for the protonated form and 316K for the non-protonated form. This may suggest a possible 1% change in the stability upon dansylation. In Figure 5C, is shown the end-to-end distance distribution of the protonated and non-protonated form the helical heteropeptide as a function of temperature, applying equation 1. This study shows a significant difference in the distance distribution in the temperature range between 273K-330K. In the protonated form, there present a decrease in the end-to-end distance with increasing temperature between 273K-313K, with a gradual increase in the distance above that temperature and throughout the spectral range. In the non-protonate form the end-to-end distribution is very different compared to the protonated form in the temperature range between 273K-330K. There is a small increase in the distance between 273-295K. There is a small decrease in the distance between 295K-330K with a gradual increase in the distance above that temperature and throughout the spectral range. The observed difference in the end-to-end distribution of these two forms of the helical heteropeptide may suggest a very different unfolding pathway with increasing temperature. In the neutral form, as it unfolds, the two ends initially move further away from each other, then a comes closer before getting further away, again. In the protonated form, on the other hand, two ends are getting closer before going further away as it unfolds as a function of increasing temperature. The dansylated system showed an increase in FRET efficiency (E) between 268 and 298 K, for the protonated form followed by a decrease in E between 298 and 363 K. In the neutral form FRET efficiency does not increase between 268 K - 298K compared to the protonated form of this system. In the course of the thermal unfolding of WH21/WH21n-dans, the donor−acceptor distance starts barely above or at 22.0 Å at 268 K and exhibits a small but statistically significant decrease to 21.5 Å at 298 K for the protonated system and nearly flat in the unprotonated system, before expanding to about 24.5 Å at the highest temperature of 363 K. We emphasize that these distances are only estimates due to the assumption of κ2 = 2/3, but most importantly here, the relative changes in distances remain significant. The unfolding pathway described by the end-to-end distance distribution is painting a different picture than by just a shifting equilibrium between the two fixed population of conformers. In the protonated form initial unfolding generates a structural population that are shorter size and in the subsequent time interval takes on a larger size. This picture is consistent with previous SAXS study28. Where they found a low RMSD structures at lower



temperatures described as broken helix although NMR and CD observed a fully helical structure. In our non-protonated system FRET not only shows no initial decrease in the end-to-end distance but also showed a slight increase or nearly flat end-to-end distance. Suggesting an absence of such broken hex in this non-protonated form in the initial stages of unfolding possibly due to increase stability due to decrease in Trp-His interaction stress.

Computational results Melting curves. The REMD melting curves for WH21 and WH21n are shown in Figure 6, showing that the non-protonated peptide clearly forms a more stable helix. The α -helix fractions at 300 K were 14-17% for WH21 and 22-23% for WH21n. These results are in quite good qualitative agreement with the experimental data. The shapes of the simulated melting curves are not as steep as the experimental ones, most probably due to properties of the TIP3P water model, which does not reproduce well the observed changes of water properties with temperature29. Trp…His distances and individual residue conformations. One significant difference between the two peptides is in the distribution of Trp…His sidechain distances, which tend to remain closer together in the protonated and further apart in the neutral form. Figure 7 shows the probability distributions of this distance in the two forms of the peptide, while the detailed MD time courses are shown in the Supplementary Information. As discussed above, the larger Trp…His separation in the neutral WH21 form is accompanied by higher overall α-helix content. Helicities of individual residues as measured by populations of the α-helix region of the Ramachandran (φ,ψ) plot and populations of individual i…i+4 hydrogen bonds are shown in Figure 8. This indicates that the higher stability of the helix in the neutral peptide is due primarily to increase of helical populations of the central residues, 6-18. Smaller helix population increases were found in the Cterminal residues 19-21, while in the N-terminus there was a small increase of helical stability for the protonated peptide. Arginine behavior. To monitor the effect of histidine deprotonation on the positively charged arginine sidechains, we have analyzed Arg sidechain conformations and the guanidinium group distances from each other and from the tryptophan and histidine sidechains. The results are shown in Figures S5-S7. Interestingly, all three arginines exhibit low but non-zero populations of close


Page 24 of 50

Page 25 of 50 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


contacts with the Trp and His sidechains in the 3-5 Å range. The Arg…Trp and Arg…His distance distributions are slightly shifted toward lower values in WH21n, with the strongest effects found for Arg9 (Figures S5-S6). The Arg…Arg distance also exhibited an analogous small shift (not shown). The arginine residues sample mostly similar conformations in the two systems (Figure S7), though with different probabilities. The largest difference is in the population of the (1, 2, 3) = g-g-t conformer, which occurs only in for Arg9 in WH21 and Arg19 in WH21n. Overall, the response of arginine locations to the deprotonated histidine is consistent with the expected effects of positive charge removal.

Ramachandran plots. The backbone conformations sampled by the peptide are shown in the Ramachandran (φ,ψ) map in Figure 9. This figure indicates that the residues sample two main conformations – the α and  regions, as well as two minor ones – αL and (61o,-173o).

Folding paths. The folding paths of the two peptides are shown in Figures 10-12. Figure 10 presents the variation of peptide volume with RMSD from helix. The data indicate that the broadest distribution of peptide volumes corresponds to intermediate values of RMSD in the 8-10 Å range, and also that the neutral peptide form has a higher population of low RMSD, i.e. folded states. Figure 11 shows the variation of number of helical hydrogen bonds with peptide shape, described by the * parameter, which changes from the value of 0 for a sphere to 1 for elongated shapes30,31 (equation provided in Supplementary Information). This figure displays several features of the peptide conformers. First, while the helix belongs in the elongated category of peptide shapes, with * of about 0.7, there is a population of more elongated conformations, both close to the helix and in the intermediate and unfolded region. Second, the distribution of shapes for intermediate and unfolded peptide structures is very wide. Finally, we again see a larger population of folded forms for the neutral peptide. Figure 12 presents a statistical analysis of hydrogen boding patterns. The horizontal axis here shows the starting residue number i for each i…i+4 helical hydrogen bond, while the vertical axis shows the total number of hydrogen bonds. For structures with 3-5 hydrogen bonds we can follow the helix nucleation process. In the neutral peptide we see a slight preference for nucleation at the N-terminus, mostly uniform nucleation tendencies in the central region 5-15



and weaker nucleation at the C-terminus. The protonated form also shows enhanced propensity for nucleation at the N-terminus and low propensity at the C-terminus, but nucleation for residues 46 is perturbed, presumably due to the strong Trp…His+ interactions. In the intermediate region, with 8-12 hydrogen bonds, the behavior of the two forms is the most different. The neutral form clearly starts full helix formation in the middle, for residues 6-13, and at a later stage, when 9-11 bonds are formed. The protonated form initiates helix formation at an off-center region, at residues 11-16, but at an earlier stage, with 7-8 hydrogen bonds formed. Similar behavior has been seen in our previous studies of WH21 with other force fields9,18. Additionally, the final stages of the neutral form helix folding involve roughly symmetric propagation from the center to both termini, while in the protonated form we see a propagation favoring the N-terminus and disfavoring the Cterminus30,31.

Global dynamics. The autocorrelation functions of the fluctuations of the number of α-helical hydrogen bonds and number of residues in α-helical region of the Ramachandran plot are shown in Figure 13. The long relaxation times, reflecting global peptide dynamics, were 360 ns for WH21 and 600-800 ns for WH21n. The estimated statistical errors of these quantities are about 25% for WH21 and 35% for WH21n. Thus, our MD simulations predict a higher helix content and slower relaxation time for WH21n. These are remarkable changes of properties for such a relatively small chemical difference. Detailed comparison of sampled structures. To compare the structures sampled by the two peptides, two approaches were used – principal component analysis (PCA) and secondary structure description. For PCA32, structures were sampled every 20 ns from each peptide trajectory and overlaid on the same reference (the WH21 ideal helix) by C RMSD. Next, the Cartesian average structures coordinates i=1,…,63 and C atom covariance matrices Cij = , i=1,…,63, j=1,…,63 were calculated. Structure sets for both peptides were then projected on the four WH21 eigenvectors with largest eigenvalues. The projections of WH21 and WH21n structures fall in the same regions of conformational space, with no visible separations of structures for either peptide. This indicates that the two peptide forms sample the same types of structures. The PCA analysis results are shown in Figure S8.


Page 26 of 50

Page 27 of 50 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


The secondary structure analysis has been performed using the program STRIDE33. MD trajectories were clustered by C RMSD with a 5 Å radius, yielding 80 structures for WH21 and 79 for WH21n. The secondary structures of the top ten clusters by population were then analyzed. The results show that the two peptides sample the same general set of conformations – partially formed helices at N- and C-termini, helix-turn-helix motifs and sections of 310 helix. The two main differences between the peptide forms are the higher population of structures with significant helix content and more helical conformers in the peptide center for WH21n relative to WH21. This is in accord with the conformational results shown in Figure 8. The secondary structure results are shown in Figures S9 and S10. Representative structures from the MD trajectory are shown in Figure 14. Generally, WH21 and WH21n tend to sample the same set of conformers but with different weights, with more helix-like conformers present in the neutral form.

Conclusion. Driven by the desire to understand how tryptophan-histidine interaction influences the overall structural stability of an -helical peptide, we have studied a twenty-one residue -helical system by creating a two different ionization states of a single histidine located four residues away from a single tryptophan in the chain sequence, with a combination of experimental measurements and comprehensive molecular dynamics simulation. The experimental component involved far UV circular dichroism spectroscopy, steady state fluorescence, and fluorescence resonance energy transfer (FRET) measurements with a neutral and a protonated histidine. We have observed that by simply removing a proton from a single histidine residue the overall stability of this -helical system increases, corresponding to a melting temperature increase by sixteen Kelvin. In the computational part of this project, we have carried out molecular dynamics and replica exchange molecular dynamics simulations of the peptide in the two ionization states. The simulations reproduce the observed effect of increased helix content and increased Trp…His separation in the neutral peptide. Additionally, the computational results suggest that the decrease in Trp…His interactions leads to specific stabilization of hydrogen bonds and helical conformations in the



center of the peptide. Analysis of the simulated folding pathway suggests that the neutral peptide has a more uniform helix nucleation and propagation propensity along the chain, while the Trp..His interactions present in the neutral form disrupt nucleation around the His position and lead to helix formation through an off-center intermediate involving residues 11-16. Finally, analysis of the rates of structural fluctuations in the molecular dynamics trajectories predicts the folding relaxation times of ca. 360 ns for the protonated and ca. 600-800 ns for the neutral form at 300 K. The first result is in excellent agreement with previous experimental data, while the second result suggests that both helix stability and kinetics are strongly influenced by the protonation state of the single histidine residue. The useful mechanistic insights from our study demonstrate the power of joint experimental-computational approaches to complex biophysical problems.

Acknowledgments. G.S.J. would like to thank W. A. Eaton for helpful discussions. We acknowledge support from XSEDE grant TG-MCB 16009 for computer time. Parts of the simulations described were conducted at the Center for Research Computing at the University of Kansas. This project was supported in part by NSF grant 180785. We also gratefully acknowledge support from the Department of Chemistry, University of Kansas General Research Fund. This article contain supplementary information that is available with this manuscript.

Figure Captions Figure 1. Far UV CD and SVD component analysis of the neutral and protonated form of histidine (His) in alpha helical heteropeptide. (A) Far UV CD of His0 WH21n from 266 to 363 K. (B) First three singular value decomposition (SVD) components (Green, Red, and Blue) of the spectra of His0 (C) Amplitude vectors of the first three components (Green, Red, Blue) of His0 form of WH21n shown as a function of temperature. (D) Far UV CD of His+ form of WH21 from 266 to 363 K. (E) First three singular value decomposition (SVD) components (Blue, Red, Green) of the spectra (F) Amplitude vectors of the first three components from SVD analysis of His+ form of WH21 shown as a function of temperature. A two-state fit with EXAM to the first component


Page 28 of 50

Page 29 of 50 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


amplitude vectors as a function of temperature yielded a Tm for His0 and His+ of a magnitude of 313K and 297K, respectively.

Figure 2. Thermal denaturation curve of WH21n/WH21 with the neutral (His0, green) and protonated (His+, blue) form of histidine. (A) Molar ellipticity as a function of temperature is presented with a fixed wavelength at 222nm. Red line is the fit to the data with a two-state model. (B) Fraction helix content of WH21n/WH21 as a function of temperature, with protonated histidine (His+) in red and neutral histidine (His0) in green.

Figure 3. Steady state fluorescence spectra of the non-dansylated W1-H5-21 peptide with protonated and neutral histidine are measured between the temperature range 268K to 363 K. SVD component analysis of these two sets of temperature dependent fluorescence spectra is carried out both with respect to wavelength and temperature. (A) fluorescence spectra of WH21 with His+ as a function wavelength are shown. Fluorescence intensity is decreasing with increasing temperature; (B) First three SVD components are shown for His+ form of the peptide; (C) First three SVD components with respect to temperature are shown with the peptide in His+ form. The amplitudes of the first components at a temperature range between 273K - 330K shows almost no change in the fluorescence intensity, suggesting a quenching process of TRP fluorescence with His+. (D) fluorescence spectra of WH21n with His0 as a function wavelength is shown. There is a change in fluorescence intensity with increasing temperature; (E) First three SVD components are shown of the peptide with a neutral histidine; (F) First three SVD components as a function of temperature of the peptide with His0 are presented. The amplitudes of the first components show a sharp decrease in the fluorescence intensity as a function of temperature with a neutral histidine in comparison with His+ form. This suggests that the TRP fluorescence is no longer quenched in the presence of a neutral histidine. Figure 4. Temperature dependent fluorescence spectra of the dansylated W1-H5-21 peptide with a His+ and a His0 are measured between the temperature range 268K - 363 K and SVD component analysis is carried out. (A) fluorescence spectra of dansylated WH21n with His0 as a function wavelength are shown. Fluorescence intensity is decreasing with increasing temperature; (B) First



three SVD components are shown for His0 form of the dansylated helical peptide; (C) First three SVD components with respect to temperature are shown of the dansylated peptide in the presence of a His0. The amplitudes of the first components show a sharp decrease in the fluorescence intensity as a function of temperature with a neutral histidine in comparison with the presence of a His+. This TRP fluorescence is no longer quenched in the presence of a neutral histidine. (D) fluorescence spectra of the dansylated-WH21 with a His+ as a function wavelength is shown. There is a change in fluorescence intensity with increasing temperature; (E) First three SVD components are shown of the peptide with in the presence of a His+. (F) First three SVD components as a function of temperature of the dansylated peptide with His+ are presented. The amplitudes of the first components at a temperature range between 273K - 330K shows almost no change in the fluorescence intensity, suggesting a quenching process of TRP fluorescence in the presence of a His+ four residue away from TRP in the -helical sequence.

Figure 5. Thermal denaturation curve of the dansylated-W1-H5-21 with the neutral (His0, green) and protonated (His+, blue) form of histidine. (A) Molar ellipticity as a function of temperature is shown with a fixed wavelength at 222nm. Red line is the fit to the data with a two-state model. (B) Shown fraction helix content of the dansylated-WH21/WH21n as a function of temperature, with a protonated histidine (His+) in blue and a neutral histidine (His0) in green. (C) End-to-End distance of the -helical heteropeptide as a function of temperature with a protonated (His+, blue) and a neutral (His0, green) histidine as a function temperature is shown. Distance between donoracceptor pair is calculated at a temperature between 268K and 363 K with FRET measurements Figure 6. Melting curves obtained from REMD with CHARMM36 force field and TIP3P water. The measures of helix content were A) the number of α-helical hydrogen bonds, counted as number of C=O(i) …H-N(i+4) contacts with O…N distances below 3.6 Å and B) the number of residues in the helical region of the Ramachandran map, counted as the number of residues within a 20o radius of the ideal helix at (φ,ψ) =(-62o,-41o). Figure 7. Distributions of (A) Trp…His sidechain distances, (B) RMSD from ideal helix and (C) end-to-end distance sampled in MD simulations of WH21 and WH21n.


Page 30 of 50

Page 31 of 50 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) Average populations of structures of WH21/WH21n in which individual residues were within 20o of the ideal helix conformation in the Ramachandran map, (φ,ψ) = (-62o,-41o) (B) Structures in which individual helical hydrogen bonds i…i+4 at residue i were below 3.6 Å in length. Data from MD trajectories. Figure 9. Distribution of backbone (φ,ψ) dihedrals from MD simulations, averaged over all peptide residues from MD trajectories. Two main regions are sampled, corresponding to α , centered at (72o,-33o) and  (-86o,143 o) structures, as well as two minor ones – αL (64o,42 o) and (61o,-173o). Distribution represented as a potential of mean force F(x,y) = -RTlnP(x,y), at T=300 K, with lowest values shown in purple, highest in red. (A) Neutral form, WH21n (B) Protonated form, WH21. Figure 10. Two-dimensional distribution of RMSD from ideal helix vs. peptide volume from MD trajectories. Distribution represented as a potential of mean force F(x,y) = -RTlnP(x,y), at T=300 K, with lowest values shown in red, highest in blue. (A) Neutral form, WH21n (B) Protonated form, WH21. Figure 11. Two-dimensional distribution of number of alpha-helical hydrogen bonds vs. peptide shape, measured by * parameter (*=0 for sphere, *=1 for elongated shapes), from MD trajectories. Distribution represented as a potential of mean force F(x,y) = -RTlnP(x,y), at T=300 K, with lowest values shown in red, highest in blue. (A) Neutral form, WH21n (B) Protonated form, WH21. Figure 12. Statistics of hydrogen bond patterns found in all replicas of the REMD simulations. Horizontal axis shows the starting residue number i of the i…i+4 hydrogen bond, the vertical axis the total number of helical hydrogen bonds formed in the structure, and the color encode the population, ranging from 0 (blue) to 1 (purple). . (A) Neutral form, WH21n (B) Protonated form, WH21. Figure 13. Autocorrelation functions C1(t) = for fluctuations in the number of α–helical hydrogen bonds and number of residues in the α-helical region of the Ramachandran map, from MD simulations. Figure 14. Representative structures sampled from MD trajectories. Shown are central structures from the five most highly populated clusters obtained by Cα atom RMSD clustering with a radius



of 5 Å. The cluster populations were: 20%, 16%, 11% , 7% and 6% for WH21 and 39%, 10%, 7%, 5% and 4% for WH21n. (A) Neutral form, WH21n (B) Protonated form, WH21.


Page 32 of 50

Page 33 of 50 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


References.

1. Kendrew, J. C.; Dickerson, R. E.; Strandberg, B. E.; Hart, R. G., Davies, D. R.; Phillips, D. C.; Shore, V. C. Structure of myoglobin: A three-dimensional Fourier synthesis at 2 Å resolution. Nature 1960, 185, 422–427. 2. Neurath, H. Intramolecular folding of polypeptide chains in relation to protein structure". J. Phys. Chem. 1940, 44, 296–305. 3. Pauling, L.; Corey, R. B.; Branson, H. R. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. PNAS, USA. 1951, 37, 205–211. 4. Marqusee, S.; Robbins, V. H.; Baldwin, R. L. Unusually stable helix formation in short alanine-based peptides. PNAS 1989, 86, 5286-5290. 5. Thompson, P. A.; Munoz, V.; Jas, G. S.; Henry, E. R.; Eaton, W. A.; Hofrichter, J. The Helix-Coil Kinetics of a Heteropeptide. J. Phys. Chem. B 2000, 104, 378−389. 6. Jas, G. S.; Eaton, W. A.; Hofrichter, J. Effect of Viscosity on the Kinetics of Alpha-Helix and Beta-Hairpin Formation. J. Phys. Chem. B 2001, 105, 261−272. 7. Jas, G. S.; Kuczera, K. Equilibrium Structure and Folding of a Helix-Forming Peptide: Circular Dichroism Measurements and Replica-Exchange Molecular Dynamics Simulations. Biophys. J. 2004, 87, 3786−3798. 8. Hegefeld, W. A.; Chen, S. E.; DeLeon, K. Y.; Kuczera, K.; , Jas, G. S. Helix formation in a pentapeptide: experiment and force-field dependent dynamics.. J. Phys Chem. A. 2010, 114(, 12391-13402. 9. Jas, G. S.; Kuczera, K. Computer simulations of helix folding in homo- and heteropeptides. Molecular Simulation 2012, 38, 682-694. 10. Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Primary structure effects on peptide group hydrogen exchange. PROTEINS Structure, Function, and Genetics 1993, 17, 75-86. 11. Chi, E. Y.; Krishnan, S.; Randolph, T. W.; Carpenter, J. F. Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces Nonnative Protein Aggregation. Pharm. Res. 2003, 20, 1325-1326. 12. Fraser, P. E.; Nguyen, J. T.; Surewicz, W. K.; Kirschner, D. E. pH-dependent structural transitions of Alzheimer amyloid peptides. Biophys. J. 1991, 60, 1190-1201. 13. Lau, S. Y.; Taneja, A. K.; Hodges R. S. Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of twostranded alpha-helical coiled-coils. J. Biol. Chem. 1984, 259, 13253-13261. 14. Blanco, F. J.; Rivas, G Serrano, L. A short linear peptide that folds into a native stable βhairpin in aqueous solution. Nat. Struc. Biol. 1994, 1, 584-590. 15. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science, 1994, 266, 776-779. 16. Pace, C. N.; Trevino, S.; Prabhskaran, E.; Scholtz, J. M. Protein structure, stability and solubility in water and other solvents. Phil. Trans. R. Soc. Lond. B 2004, 359, 1225–1235. 17. Kirchhoff, W. H.Exam (Coden: Ntnoef). U.S. Government Printing Office: Washington, DC, 1993; Vol. 1401.



18. Jas, G. S.; Hegefeld, W. A.; Russel, C. R..; Johnson, C. K..; Kuczera, K.; Detailed Microscopic Unfolding Pathways of an α‑Helix and a β‑Hairpin: Direct Observation and Molecular Dynamics. J. Phys. Chem. B 2014, 118, 7233−7246. 19. (a) Schuler, B.; Eaton, W. A. Protein Folding Studied by SingleMolecule FRET. Curr. Opin. Struct. Biol. 2008, 18, 16−26. (b) Gopich, I. V.; Szabo, A. Theory of the Energy Transfer Efficiency and Fluorescence Lifetime Distribution in Single-Molecule FRET. PNAS, U.S.A. 2012, 109, 7747−7752. 20. Overton, M. C.; Blumer, K. J. Use of Fluorescence Resonance Energy Transfer to Analyze Oligomerization of G-Protein-Coupled Receptors Expressed in Yeast. Methods 2002, 27, 324−332. 21. Howlett, D. R. Misfolding in Disease: Cause or Response? Curr. Med. Chem.: Immunol., Endocr. Metab. Agents 2003, 3, 371−383. 22. Brooks, B. R.; Brooks, C. L.; Mackerell, A. D.; Nilsson, L.; Petrella, R. J.; Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S.; et al. CHARMM: The biomolecular simulation program. J. Comput. Chem. 2009, 30, 1545−1614. 23. Jorgensen, W.L, Chandrasekhar, J., Madura, J.D., Impey, R.W., Klein, M.L. Comparison of simple potential functions for simulating liquid water. J Chem Phys 1983, 79, 926-935. 24. Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 2008, 4, 435−447. 25. MacKerell, Jr., A. D.; Bashford, D.; Bellott, M.; Dunbrack Jr., R.L.;Evanseck, J.D.; Field, M.J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F.T.K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D.T.; Prodhom, B.; Reiher, III, W.E.; Roux, B.; Schlenkrich, M.; Smith, J.C.; Stote, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M. All-atom empirical potential for molecular modeling and dynamics Studies of proteins. J. of Phys. Chem. B, 1998, 102, 3586-3616. 26. Chung, H. S.; Gopich, I. V.; McHale, K.; Cellmer, T.; Louis, J. M.; Eaton, W. A. Extracting Rate Coefficients from Single-Molecule Photon Trajectories and FRET Efficiency Histograms for a Fast-Folding Protein. J. Phys. Chem. A 2011, 115, 3642–3656. 27. Chung, H. S.; Eaton, W. A. Protein folding transition path times from single molecule FRET. Curr Opin Struct Biol. 2018, 48, 30-39. 28. Zagrovic, B.; Jayachandran, G.; Millett, I. S.; Doniach, S.; Pande, V. S. How Large Is an Alpha-Helix? Studies of the Radii of Gyration of Helical Peptides by Small-Angle X-Ray Scattering and Molecular Dynamics. J. Mol. Biol. 2005, 353 (2), 232−241. 29. Jorgensen, W.L., Jenson, C. Temperature dependence of TIP3P, SPC and TIP4P water from NPT Monte Carlo simulations: Seeking temperatures of maximum density. J. Comp. Chem. 1998, 19, 1179-1186. 30. Steinhauser, M. O. A molecular dynamics study of universal properties of polymer chains in different solvent qualities. Part I. A review of linear chain properties. J. Chem. Phys. 2005, 122, 094901. 31. Tran, M.T., Mao, A., Pappu, R.V. Role of backbone-solvent interactions in determining conformational equilibria of intrinsically disordered proteins. J. Am. Chem. Soc. 2008, 130, 7380-7392.


Page 34 of 50

Page 35 of 50 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


32. David, C.D. , Jacobs, D.J. Principal component analysis: A method for determining the essential dynamics of proteins. Methods Mol Biol 2014, 1084, 183-226. 33. Frishman, D., Argos, P. Knowledge-based protein secondary structure assignment. Proteins 1995, 23, 566-579.



Figure 1.


Page 36 of 50

Page 37 of 50 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.



Figure 3.


Page 38 of 50

Page 39 of 50 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.



Figure 5.

Figure 6.


Page 40 of 50

Page 41 of 50 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 7.



Figure 8.


Page 42 of 50

Page 43 of 50 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.



Figure 10.


Page 44 of 50

Page 45 of 50 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 11.



Figure 12.


Page 46 of 50

Page 47 of 50 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 13.



Figure 14.


Page 48 of 50

Page 49 of 50 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


Table 1. Thermodynamics parameters for Folded -> unfolded conformation.

NonDansylated

NonDansylated

Dansylated

Dansylated

pH4.8

pH7.2

pH4.8

pH7.2

297

313

303

316

52.4

48.5

57

54

0.18

0.15

0.19

0.17

-0.044

0.54

0.26

0.83

1.08

0.40

0.65

0.25

0.48

0.70

0.61

0.80

Tm (K)

H0 (Kcal/mol) S (Kcal/mol.K) 0

G 298 (Kcal/mol.K) 0

K298

Fraction Helix298




Page 50 of 50

Deprotonation of a Single Amino Acid Residue Induces Significant

Recommend Documents