Effects of denaturants on the dynamics of loop formation in polypeptides

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.     

Kinetics of Contact Formation and End-to-End Distance Distributions of Swollen Disordered Peptides

Unstructured polypeptide chains are subject to various degrees of swelling or compaction depending on the combination of solvent condition and amino acid sequence. Highly denatured proteins generally behave like random-coils with excluded volume repulsion, whereas in aqueous buffer more compact conformations have been observed for the low-populated unfolded state of globular proteins as well as for naturally disordered sequences. To quantitatively account for the different mechanisms inducing the swelling of polypeptides, we have examined three 14-residues peptides in aqueous buffer and in denaturant solutions, including the well characterized AGQ repeat as a reference and two variants, in which we have successively introduced charged side chains and removed the glycines. Quenching of the triplet state of tryptophan by close contact with cysteine has been used in conjunction with Förster resonance energy transfer to study the equilibrium and kinetic properties of the peptide chains. The experiments enable accessing end-to-end root mean-square distance, probability of end-to-end contact formation and intrachain diffusion coefficient. The data can be coherently interpreted on the basis of a simple chain model with backbone angles obtained from a library of coil segments of proteins and hard sphere repulsion at each C_α position. In buffered water, we find that introducing charges in a glycine-rich sequence induces a mild chain swelling and a significant speed-up of the intrachain dynamics, whereas the removal of the glycines results in almost a two-fold increase of the chain volume and a drastic slowing down. In denaturants we observe a pronounced swelling of all the chains, with significant differences between the effect of urea and guanidinium chloride.     

Kinetics of internal-loop formation in polypeptide chains: a simulation study

The speed of simple diffusional motions, such as the formation of loops in the polypeptide chain, places one physical limit on the speed of protein folding. Many experimental studies have explored the kinetics of formation of end-to-end loops in polypeptide chains; however, protein folding more often requires the formation of contacts between interior points on the chain. One expects that, for loops of fixed contour length, interior loops will form more slowly than end-to-end loops, owing to the additional excluded volume associated with the "tails". We estimate the magnitude of this effect by generating ensembles of randomly coiled, freely jointed chains, and then using the theory of Szabo, Schulten, and Schulten to calculate the corresponding contact formation rates for these ensembles. Adding just a few residues, to convert an end-to-end loop to an internal loop, sharply decreases the contact rate. Surprisingly, the relative change in rate increases for a longer loop; sufficiently long tails, however, actually reverse the effect and accelerate loop formation slightly. Our results show that excluded volume effects in real, full-length polypeptides may cause the rates of loop formation during folding to depart significantly from the values derived from recent loop-formation experiments on short peptides.     

Direct computation of long time processes in peptides and proteins: reaction path study of the coil-to-helix transition in polyalanine.

The MaxFlux reaction path algorithm was used to isolate optimal transition pathways for the coil-to-helix transition in polyalanine. Eighteen transition pathways, each connecting one random coil configuration with an ideal alpha-helical configuration, were computed and analyzed. The transition pathway energetics and mechanism were analyzed in terms of the progression of the peptide nonbonded contact formation, helicity, end-to-end distance and energetics. It was found that (1) localized turns characterized by i, i + 3 hydrogen bonds form in the early stages of the coil-to-helix transition, (2) the peptide first collapses and then becomes somewhat more extended in the final stage of helix formation, and (3) 310-helix formation does not appear to be a necessary step in the transition from coil to helix. These conclusions are in agreement with the results of more computationally intensive direct molecular dynamics simulations. Proteins 1999;36:249-261.     

Reinterpretation of GCN4-p1 folding kinetics: partial helix formation precedes dimerization in coiled coil folding.

The folding of coiled coil peptides has traditionally been interpreted in terms of native dimer and unfolded monomers. Calculations using AGADIR and experimental studies of fragments suggest that the monomers of the coiled coil peptide, GCN4-p1, contain significant residual helical structure. A simple model based on diffusion-collision theory predicts not only the measured folding rate within an order of magnitude, but also predicts remarkably well the effect of alanine to glyXcine mutations. We suggest that intrinsic helix stability is a major determinant of the folding rate of the GCN4 coiled coil.     

Rate of loop formation in peptides: a simulation study

Experimental techniques with high temporal and spatial resolution extend our knowledge of how biological macromolecules self-organise and function. Here, we provide an illustration of the convergence between simulation and experiment made possible by techniques such as triplet-triplet energy transfer and fluorescence quenching with long-lifetime and fast-quenching fluorescent probes. These techniques have recently been used to determine the average time needed for two residues in a peptide or protein segment to form a contact. The timescale of this process is accessible to computer simulation, providing a microscopic interpretation of the data and yielding new insight into the disordered state of proteins. Conversely, such experimental data also provide a test of the validity of alternative choices for the molecular models used in simulations, indicating their possible deficiencies. We carried out simulations of peptides of various composition and length using several models. End-to-end contact formation rates and their dependence on peptide length agree with experimental estimates for some sequences and some force fields but not for others. The deviations are due to artefactual structuring of some peptides, which is not observed when an atomistic model for the solvation water is used. Simulations show that the observed experimental rates are compatible with considerably different distributions of the end-to-end distance; for realistic models, these are never Gaussian but indicative of a rugged energy landscape.     

Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study

2,2,2-Trifluoroethanol (TFE) is widely used to induce helix formation in peptides and proteins, but the mechanism behind this effect is still poorly understood. Several recent papers have proposed that TFE acts by selectively desolvating the peptide backbone groups of the helix state. Infrared (IR) spectroscopy of the amide I band of polypeptides can be used to probe both secondary structure and backbone solvation, making this technique well suited for addressing the effect of TFE on polypeptide conformation. In this paper, we report the IR spectra as a function of TFE concentration for an alanine-rich peptide based on the repeat (AAKAA)(n)(). The IR spectra confirm that TFE desolvates the helical state of the peptide to a greater extent than the random coil state. Moreover, using a series of specifically (13)C-labeled peptides, the precise residues desolvated in the presence of TFE were identified. The residues most desolvated by TFE are the alanines located at position i - 4 in the sequence, where i is a lysine residue. This pattern of desolvation is consistent with molecular dynamics simulations which predict strong interactions between the lysine side chain at position n and the backbone carbonyl of the alanine at position i - 4. This is the first direct spectroscopic evidence of specific desolvation of helix backbone atoms in model alanine-rich peptides.     

Equilibrium unfolding of the poly(glutamic acid)20 helix

The equilibrium structural ensemble of a 20-residue polyglutamic acid peptide (E(20)) was studied with FRET, circular dichroism, and molecular dynamics (MD) simulations. A FRET donor, o-aminobenzamide, and acceptor, 3-nitrotyrosine, were introduced at the N- and C-termini, respectively. Circular dichroism, steady state FRET, and time-resolved FRET measurements were employed to characterize the fraction helix and end-to-end distance under different pH conditions: pH 4 (60% alpha-helix), pH 6 (0% alpha-helix), and pH 9 (0% alpha-helix). At pH 4, the end-to-end distance was measured at 24 A and determined to be considerably less than the 31 A predicted for an alpha-helix of the same length. At pH 6 and 9, the end-to-end distance was measured at > 31 and 39 A respectively, both which are determined to be considerably greater than the 27 A predicted for a freely jointed random coil of the same length. To better understand the physical forces underlying the unusual helix-coil transition in this peptide, three theoretical MD models of E(20) were constructed: (1) a pure alpha-helix, (2) an alpha-helix with equivalent attractive intramolecular contacts, and (3) a weak alpha-helix with termini-weighted intramolecular contacts ("sticky ends"). Using MD simulations, the bent helix structure calculated from Model 3 was found to be the closest in agreement with the experimental data.     

Positional independence and additivity of amino acid replacements on helix stability in monomeric peptides

The 17-residue peptide acetylAEAAAKEAAAKEAAAKAamide, described as an autonomous folding unit (Marqusee & Baldwin, 1987), has been used to examine the effect of amino acid replacements on helix stability. Alanine residues(s) at positions 4, 9, and 14 in the peptide sequence were replaced either singly or multiply by either serine or methionine residues with solid-phase peptide synthesis. The thermal dependence of the helix/coil transition of each peptide was observed by far-ultraviolet circular dichroism. Within experimental variation, all three single replacements exhibit a common thermal transition, and all three double replacements exhibit a different common thermal transition. These results suggest that replacement of the central alanine residue in the repeat EAAAK located in the N-terminus, in the middle, or in the C-terminus of the peptide helix has the same effect on helix stability. The melting temperature of each thermal transition was estimated by assuming a linear van't Hoff plot and a change in molar ellipticity of 33,500 deg cm2 dmol-1. Such analysis indicates that each replacement of an alanine residue by a serine residue diminishes the melting temperature by 11 +/- 1 degrees C and that each replacement of an alanine residue by a methionine residue diminishes the melting temperature by 6 +/- 1 degrees C. These results suggest that the effect of these replacements on helix stability is additive.     

Kinetics of intramolecular contact formation in a denatured protein

Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein ( approximately 10(4)s(-1)). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule F?rster resonance energy transfer (FRET) experiments.     

Proline for alanine substitutions in the C-peptide helix of ribonuclease A

The effect on overall alpha-helix content of substituting proline for alanine has been determined at 5 positions (1, 2, 4, 5, and 13) of a 13-residue peptide related in sequence to residues 1-13 of ribonuclease A. The helix content falls off rapidly as proline is moved inward, and the proline residue effectively truncates the helix. No helix-stabilizing effect of proline is found at positions 2 or 4 within the first turn of the helix. Proline substitution at either end position (1, 13) has little effect on overall helix content, in agreement with an earlier study of glycine for alanine substitutions. The two end residues of the helix appear to be strongly frayed.     

Nuclear magnetic resonance investigation of the helix in random coil transformation in poly-alpha-amino acids. I

High-resolution nuclear magnetic resonance spectra at 100 MHz and 220 MHz have been obtained on two samples of poly-L -alanine of differing molecular weights (2500 and 42 500) in the chloroform–trifluoroacetic acid system under various conditions of solvent composition, temperature, and polypeptide concentration. Separate helix and random coil peaks are observed for the α-CH and peptide NH backbone proton resonances, thereby permitting the determination of helix content. This observation of separate peaks demonstrates that the lifetimes of the helix and random coil portions of poly-L -alanine have lower limits of about 10^?1 sec. It is suggested that solvent–peptide versus peptide–peptide hydrogen bond competition, coupled with a destabilizing effect of the trifluoroacetic acid on the helix, is responsible for the helix–random coil transformation.     

The interplay between transient α-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts

The peptide backbones of disordered proteins are routinely characterized by NMR with respect to transient structure and dynamics. Little experimental information is, however, available about the side chain conformations and how structure in the backbone affects the side chains. Methyl chemical shifts can in principle report the conformations of aliphatic side chains in disordered proteins and in order to examine this two model systems were chosen: the acid denatured state of acyl-CoA binding protein (ACBP) and the intrinsically disordered activation domain of the activator for thyroid hormone and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the γ-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation of γ-gauche effect. To overcome this, we reference the chemical shifts to those in a more disordered state resulting in residue specific random coil chemical shifts. The (13)C secondary chemical shifts of the methyl groups of valine, leucine, and isoleucine show sequence specific effects, which allow a quantitative analysis of the ensemble of χ(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly or indirectly related to formation of transient α-helices.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of α-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining α-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The α-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each α-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set I] and [set 2]) and for 30 mostly α-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix—coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix—coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area. © 1997 John Wiley & Sons, Inc. Biopoly 42: 239–269, 1997     

Vimentin Coil 1A—A Molecular Switch Involved in the Initiation of Filament Elongation

Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric α-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T_m). At a concentration of 0.1 mg/ml, the T_m of the peptide with the single point mutation Y117L increased dramatically by 46 °C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by X-ray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might “switch” from a dimeric α-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction.     

Molecular dynamics study of structure and stability of a model coiled coil

This paper employs methods used earlier to study helix propensity in a model α-helix. The methods are extended to simulations of a motif structure of the α-helical coiled coil, i.e., a structure with a simple amino acid sequence, containing only alanine, leucine, and valine, with leucine and valine forming hydrophobic contacts in the helix interface (positions “d” and “a”). Dynamic simulations of the model coiled-coil structure reproduce characteristic features of the coiled-coil motif seen in experimental studies. Free energy simulations were used to assess the change in stability of the model when a leucine pair or a valine pair in the helix interface was replaced with an alanine pair. A leucine pair at position d was found to contribute 3.4 kcal/mol to the stability of the coiled coil relative to an alanine pair, and a valine pair at postion a was found to contribute 0.8 kcal/mol relative to an alanine pair. The value for the leucine pair agrees with reports in two experimental studies with molecules having different amino sequence. The value for the valine pair is reasonable given the smaller size of the valine side chain and the intrinsic low helix propensity of valine. No experimental value was available for comparison. © 1993 Wiley-Liss, Inc.     

Design, synthesis and structure of an amphipathic peptide with pH-inducible haemolytic activity.

A synthetic, 26-residue peptide having a strong helix forming potential in the protonated state was designed to interact with lipid bilayers in a pH-dependent way. On the basis of this concept a cluster of four glutamic acid residues was inserted in the central region of the amphipathic peptide to promote helix destabilization by mutual charge repulsion at neutral pH. Protonation of these residues might then bring about both a pH-mediated change in hydrophobicity and conformation forming a membrane-active amphiphilic helix. The sequence GLGTLLTLLEFLLEELLEFLKRKRQQamide produced by the design strategy induced pH-triggered lysis of human erythrocytes. A molecular model correlating the lytic activity to the formation of transmembrane pores which were detected by electron microscopy in erythrocyte membranes is discussed. Circular dichroism studies indicated a self-association of the monomeric random coil form with increasing peptide concentration leading to the apparent induction of strong alpha-helix formation (approximately 100% helicity) in the fully aggregated state. However, no pH-dependent helix-random coil transition was observed, implying that interhelical hydrophobic and ionic interactions not only govern the self-association but also decisively influence the conformational stability of the peptide.     

Stability of the helical conformation of random L-alanine-L-lysine copolymers in aqueous solution

The potentiometric titration of random copolymers of L -lysine and L -alanine containing 0–35% alanine was carried out. The standard free-energy change for the transition of coil to helix was calculated from the titration curve, and was treated by taking account of first-neighbor interactions. For uncharged lysine ΔG° = ?140 cal/mole, and for alanine ΔG° = ?50 cal/mole in 0.06M NaBr at 25°C, indicating that the alanine helix is thermodynamically less stable than the lysine helix. The randomness in co-polymerization was confirmed by trypsin treatment.