Daniel Verbaro

The pH Dependence of the 695 nm Charge Transfer Band Reveals the Population of an Intermediate State of the Alkaline Transition of Ferricytochrome c at Low Ion Concentrations

We have measured and analyzed the pH dependence of the 695 nm charge transfer band of horse heart ferricytochrome c as a function of pH between 7.0 and 10.5 at high (50 mM) and low (0.5 mM) phosphate ion concentrations. Our data clearly reveal that the transition from the native state (III) to the two alkaline states (IV) involves two deprotonation steps which cannot be assigned to the two different lysine ligands associated with the two alkaline states. While the respective pK values are rather similar at high phosphate concentrations (9.23 and 9.14), they are clearly different at low anion concentrations (9.65 and 8.5). Apparently, the deprotonation that can be assigned to a pK of 8.5 populates an intermediate state termed III*, in which M80 is still an axial ligand. A comparison of Soret band CD spectra suggests that III* bears some similarity with the recently characterized thermally excited state IIIh. Our data suggest that the current picture of the alkaline transition is incomplete. The obtained results might be of relevance for characterizing the structure of ferricytochrome c bound to anionic phospholipids.

Role of enthalpy-entropy compensation interactions in determining the conformational propensities of amino acid residues in unfolded peptides.

The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.

Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. II. Tripeptides with short side chains populating asx and β‐type like turn conformations

In the preceding paper, we found that ensembles of tripeptides with long or bulky chains can include up to 20% of various turns. Here, we determine the structural and thermodynamic characteristics of GxG peptides with short polar and/or ionizable central residues (D, N, C), whose conformational distributions exhibit higher than average percentage (>20%) of turn conformations. To probe the side‐chain conformations of these peptides, we determined the 3J(Hα,Hβ) coupling constants and derived the population of three rotamers with χ1‐angles of −60°, 180° and 60°, which were correlated with residue propensities by DFT‐calculations. For protonated GDG, the rotamer distribution provides additional evidence for asx‐turns. A comparison of vibrational spectra and NMR coupling constants of protonated GDG, ionized GDG, and the protonated aspartic acid dipeptide revealed that side chain protonation increases the pPII content at the expense of turn populations. The charged terminal groups, however, have negligible influence on the conformational properties of the central residue. Like protonated GDG, cationic GCG samples asx‐turns to a significant extent. The temperature dependence of the UVCD spectra and 3J(HNHα) constants suggest that the turn populations of GDG and GNG are practically temperature‐independent, indicating enthalpic and entropic stabilization. The temperature‐independent J‐coupling and UVCD spectra of GNG require a three‐state model. Our results indicate that short side chains with hydrogen bonding capability in GxG segments of proteins may serve as hinge regions for establishing compact structures of unfolded proteins and peptides. Proteins 2013.

Conformational Stability of Cytochrome c Probed by Optical Spectroscopy

Over the last 50 years cytochrome c has been used as a model system for studying electron transfer and protein folding processes. Recently, convincing evidence has been provided that this protein is also involved in other biological processes such as the apoptosis and α-synuclein aggregation. Numerous lines of evidence suggest that the diversity of the functional properties of cytochrome c is linked to its conformational plasticity. This chapter introduces circular dichroism and absorption spectroscopy, as an ideal tool to explore this proteins conformational in solution. Besides assisting in distinguishing different conformations and in obtaining the equilibrium thermodynamics of the transitions between them, the two spectroscopies can also be used to explore details of heme-protein interaction, for example, the influence of the external electric field on the prosthetic heme group.

Structural Analysis of Unfolded Peptides by Raman Spectroscopy

Raman spectroscopy has positioned itself as an invaluable tool in the study of complex biological systems, consistently being used to obtain information illustrating a vast array of fundamental properties. Of primary interest, with respect to the focus of this chapter, are conformational changes of peptide backbones. For short peptides to larger biological systems this understanding can be extended to local hydrogen bonding interactions and the probing of other structural or organizational properties. With regard to unfolded peptides Raman spectroscopy can be used as a technique complementary to infrared (IR) and vibrational circular dichroism (VCD) spectroscopy. This chapter describes how high quality polarized Raman spectra of peptide can be recorded with a Raman microspectrometer and how the structure sensitive amide I band profiles of isotropic and anisotropic Raman scattering can be analyzed in conjunction with the respective IR and VCD profiles to obtain conformational distributions of short unfolded peptides.

Conformational Discrepancies Between Molecular Dynamics Force Fields and Vibrational Spectroscopy in Short Alanine-Based Peptides

Structural preferences in the unfolded state of peptides determined by molecular dynamics still contradict experimental data. A remedy in this regard has been suggested by MD simulations with an optimized Amber force field ff03∗ (R. Best and G. Hummer, J. Phys. Chem. B 113, 9004-9015). The simulations yielded a statistical coil distribution for alanine, which is at variance with recent experimental results. To check the validity of this distribution, we investigated the peptide H-A5W-OH, which with the exception of the additional terminal tryptophan is analogous to the peptide used to optimize the force field. Electronic circular dichroism, vibrational circular dichroism, infrared spectroscopy as well as J-coupling constants obtained from NMR experiments were used to derive the peptides conformational ensemble. Qualitatively, the experimental 3J(HN,Cα), VCD, and ECD indicated a preference of alanine for polyproline II-like conformations. Additionally, Forster-resonance-energy transfer between the terminal fluorophores of another analogous peptide Dbo-A5W-OH was used to determine its average length. In order to check whether the above statistical coil distribution quantitatively accounts for experimental data, we employed an excitonic model to calculate the amide I’ profiles of the IR and VCD spectrum of H-A5W-OH as well as the distance between the two terminal peptide carbonyls by using the distribution obtained from ff03∗. This led to an underestimated negative VCD couplet and an overestimated distance between terminal carbonyl groups. A better representation of experimental data was desired so we changed the distribution parameters in line with results recently obtained for alanine in GAG. This distribution model satisfactorily reproduced the amide I’ profiles, the J-coupling constant and the end-to-end distance of H-A5W-OH, which reinforces alanines high structural preference for polyproline II.

Structure Analysis of Unfolded Peptides I: Vibrational Circular Dichroism Spectroscopy

Vibrational circular dichroism (VCD) spectroscopy is an invaluable spectroscopic techniques utilized to exploit the optical strength of vibrational transitions for structure analysis. In this chapter, we describe the protocol for measuring and self-consistently analyzing VCD and the corresponding FT-IR spectra of short peptides. This process involves the decomposition of the IR spectrum as well as simulations of the amide I band profiles in both spectra based on structural models of the peptides investigated. This type of spectral analysis should be complemented with similar investigations of Raman spectra, which are described in the subsequent chapter. The structural analysis of short, unfolded peptides described in this chapter can easily be extended for the analysis of longer unfolded peptides or even proteins. This is particularly important in view of the demonstrated biological relevance of intrinsically disordered peptides and proteins (IDPs).