Tuning the thermostability of GHG gels by salts at different positions on the Hofmeister scale.

The influence of Hofmeister cations (NH4+, Na+, Mg2+) and anions (H2PO4-, CH3COO-, Cl-, NO3-) on the thermostability of a GHG hydrogel was investigated. The combined results of UV circular dichroism (UVCD) and Small Amplitude Oscillatory Shear Rheology experiments reveal that the addition of salt reduces the stability of the gel phase and the underlying fibrils. In line with the cationic Hofmeister hierarchy, the chaotropic Mg2+ ions caused the greatest thermal destabilization of the gel phase with the gel → sol transition temperature Tgs value lowered by 10 °C. In the absence of salt, the gel → sol transition probed by the storage modulus and microscopy is biphasic. In the presence of salt, it becomes monophasic. Contrary to expectations the presence of Hofmeister anions leads to a nearly identical reduction of the gel → sol transition temperatures. However, UVCD spectra suggest that they affect the ππ-stacking between imidazole groups to a different extent. We relate the absence of ion specificity regarding the solubility of fibrils (probed by UVCD) to the observed enthalpy-entropy compensation of the dissolution process. Our results combined show how CD spectroscopy and rheology combined yields a more nuanced picture of the processes underlying the gel → sol transition.

Intrinsic Conformational Dynamics of Glycine and Alanine in Polarizable Molecular Dynamics Force Fields: Comparison to Spectroscopic Data.

Molecular dynamics (MD) is a great tool for elucidating conformational dynamics of proteins and peptides in water at the atomistic level that often surpasses the level of detail available experimentally. Structure predictions, however, are limited by the accuracy of the underlying MD force field. This limitation is particularly stark in the case of intrinsically disordered peptides and proteins, which are characterized by solvent-accessible and disordered peptide regions and domains. Recent studies show that most additive MD force fields, including CHARMM36m, do not reproduce the intrinsic conformational distributions of guest amino acid residues x in cationic GxG peptides in water in line with experimental data. Positing that a lack of polarizability in additive MD force fields may be the culprit for the reported discrepancies, we here examine the conformational dynamics of guest glycine and alanine residues in cationic GxG peptides in water using two polarizable MD force fields, CHARMM Drude and AMOEBA. Our results indicate that while AMOEBA captures the experimental data better than CHARMM Drude, neither of the two polarizable force fields offers an improvement of the Ramachandran distributions of glycine and alanine residues in cationic GGG and GAG peptides, respectively, over CHARMM36m.

Probing the versatility of cytochrome c by spectroscopic means: A Laudatio on resonance Raman spectroscopy.

Over the last 50 years resonance Raman spectroscopy has become an invaluable tool for the exploration of chromophores in biological macromolecules. Among them, heme proteins and metal complexes have attracted considerable attention. This interest results from the fact that resonance Raman spectroscopy probes the vibrational dynamics of these chromophores without direct interference from the surrounding. However, the indirect influence via through-bond and through-space chromophore-protein interactions can be conveniently probed and analyzed. This review article illustrates this point by focusing on class 1 cytochrome c, a comparatively simple heme protein generally known as electron carrier in mitochondria. The article demonstrates how through selective excitation of resonance Raman active modes information about the ligation, the redox state and the spin state of the heme iron can be obtained from band positions in the Raman spectra. The investigation of intensities and depolarization ratios emerged as tools for the analysis of in-plane and out-of-plane deformations of the heme macrocycle. The article further shows how resonance Raman spectroscopy was used to characterize partially unfolded states of oxidized cytochrome c. Finally, it describes its use for exploring structural changes due to the protein's binding to anionic surfaces like cardiolipin containing membranes.

Conformational Manifold Sampled by Two Short Linear Motif Segments Probed by Circular Dichroism, Vibrational, and Nuclear Magnetic Resonance Spectroscopy.

Disordered protein segments called short linear motifs (SLiM) serve as recognition sites for a variety of biological processes and act as targeting signals, modification, and ligand binding sites. While SLiMs do not adopt one of the known regular secondary structures, the conformational distribution might still reflect the structural propensities of their amino acid residues and possible interactions between them. In the past, conformational analyses of short peptides provided compelling evidence for the notion that individual residues are less conformationally flexible than locally expected for a random coil. Here, we combined various spectroscopies (NMR, IR, vibrational, and UV circular dichroism) to determine the Ramachandran plots of two SLiM motifs, i.e., GRRDSG and GRRTSG. They are two representatives of RxxS motifs that are capable of being phosphorylated by protein kinase A, an enzyme that plays a fundamental role in a variety of biological processes. Our results reveal that the nearest and non-nearest interactions between residues cause redistributions between polyproline II and ß-strand basins while concomitantly stabilizing extended relative to turn-forming and helical structures. They also cause shifts in basin positions. With increasing temperature, ß-strand populations become more populated at the expense of polyproline II. While molecular dynamics simulations with Amber ff14SB and CHARMM 36m force fields indicate residue-residue interactions, they do not account for the observed structural changes.

The relevance of short peptides for an understanding of unfolded and intrinsically disordered proteins.

Over the last thirty years the unfolded state of proteins has attracted considerable interest owing to the discovery of intrinsically disordered proteins which perform a plethora of functions despite resembling unfolded proteins to a significant extent. Research on both, unfolded and disordered proteins has revealed that their conformational properties can deviate locally from random coil behavior. In this context results from work on short oligopeptides suggest that individual amino acid residues sample the sterically allowed fraction of the Ramachandran plot to a different extent. Alanine has been found to exhibit a peculiarity in that it has a very high propensity for adopting polyproline II like conformations. This Perspectives article reviews work on short peptides aimed at exploring the Ramachandran distributions of amino acid residues in different contexts with experimental and computational means. Based on the thus provided overview the article discussed to what extent short peptides can serve as tools for exploring unfolded and disordered proteins and as benchmarks for the development of a molecular dynamics force field.

Influence of central sidechain on self-assembly of glycine-x-glycine peptides.

Low molecular weight gelators (LMWGs) are the subject of intense research for a range of biomedical and engineering applications. Peptides are a special class of LMWG, which offer infinite sequence possibilities and, therefore, engineered properties. This work examines the propensity of the GxG peptide family, where x denotes a guest residue, to self-assemble into fibril networks via changes in pH and ethanol concentration. These triggers for gelation are motivated by recent work on GHG and GAG, which unexpectedly self-assemble into centimeter long fibril networks with unique rheological properties. The propensity of GxG peptides to self-assemble, and the physical and chemical properties of the self-assembled structures are characterized by microscopy, spectroscopy, rheology, and X-ray diffraction. Interestingly, we show that the number, length, size, and morphology of the crystalline self-assembled aggregates depend significantly on the x-residue chemistry and the solution conditions, i.e. pH, temperature, peptide concentration, etc. The different x-residues allow us to probe the importance of different peptide interactions, e.g. π-π stacking, hydrogen bonding, and hydrophobicity, on the formation of fibrils. We conclude that fibril formation requires π-π stacking interactions in pure water, while hydrogen bonding can form fibrils in the presence of ethanol-water solutions. These results validate and support theoretical arguments on the propensity for self-assembly and leads to a better understanding of the relationship between peptide chemistry and fibril self-assembly. Overall, GxG peptides constitute a unique family of peptides, whose characterization will aid in advancing our understanding of self-assembly driving forces for fibril formation in peptide systems.

Heme-Protein Interactions and Functional Relevant Heme Deformations: The Cytochrome c Case.

Heme proteins are known to perform a plethora of biologically important functions. This article reviews work that has been conducted on various class I cytochrome c proteins over a period of nearly 50 years. The article focuses on the relevance of symmetry-lowering heme-protein interactions that affect the function of the electron transfer protein cytochrome c. The article provides an overview of various, mostly spectroscopic studies that explored the electronic structure of the heme group in these proteins and how it is affected by symmetry-lowering deformations. In addition to discussing a large variety of spectroscopic studies, the article provides a theoretical framework that should enable a comprehensive understanding of the physical chemistry that underlies the function not only of cytochrome c but of all heme proteins.

Forbidden Secondary Structures Found in Gel-Forming Fibrils of Glycylphenylalanylglycine.

The zwitterionic l-tripeptide glycylphenylalanylglycine self-assembles into very long crystalline fibrils in an aqueous solution, which causes the formation of an exceptionally strong gel phase (G' ∼ 5 × 106 Pa). The Rietveld refinement analysis of its powder X-ray diffraction (PXRD) pattern reveals a unit cell with four peptides forming a P212121 space group and adopting an inverse polyproline II conformation, that is, a right-handed helical structure that occupies the "forbidden" region of the Ramachandran plot. This unusual structure is stabilized by a plethora of intermolecular interactions facilitated by the large number of different functional groups of the unblocked tripeptide. Comparisons of simulated and experimental Fourier transform infrared and vibrational circular dichroism (VCD) amide I' profiles corroborate the PXRD structure. Our experimental setup reduces the sample to a quasi-two-dimensional network of fibrils. We exploited the influence of this reduced dimensionality on the amide I VCD to identify the main fibril axis. We demonstrate that PXRD, vibrational spectroscopy, and amide I simulations provide a powerful toolset for secondary structure and fibril axis determination.

Exploring Nearest Neighbor Interactions and Their Influence on the Gibbs Energy Landscape of Unfolded Proteins and Peptides.

The Flory isolated pair hypothesis (IPH) is one of the corner stones of the random coil model, which is generally invoked to describe the conformational dynamics of unfolded and intrinsically disordered proteins (IDPs). It stipulates, that individual residues sample the entire sterically allowed space of the Ramachandran plot without exhibiting any correlations with the conformational dynamics of its neighbors. However, multiple lines of computational, bioinformatic and experimental evidence suggest that nearest neighbors have a significant influence on the conformational sampling of amino acid residues. This implies that the conformational entropy of unfolded polypeptides and proteins is much less than one would expect based on the Ramachandran plots of individual residues. A further implication is that the Gibbs energies of residues in unfolded proteins or polypeptides are not additive. This review provides an overview of what is currently known and what has yet to be explored regarding nearest neighbor interactions in unfolded proteins.

Randomizing of Oligopeptide Conformations by Nearest Neighbor Interactions between Amino Acid Residues.

Flory's random coil model assumes that conformational fluctuations of amino acid residues in unfolded poly(oligo)peptides and proteins are uncorrelated (isolated pair hypothesis, IPH). This implies that conformational energies, entropies and solvation free energies are all additive. Nearly 25 years ago, analyses of coil libraries cast some doubt on this notion, in that they revealed that aromatic, but also ß-branched side chains, could change the 3J(HNHCα) coupling of their neighbors. Since then, multiple bioinformatical, computational and experimental studies have revealed that conformational propensities of amino acids in unfolded peptides and proteins depend on their nearest neighbors. We used recently reported and newly obtained Ramachandran plots of tetra- and pentapeptides with non-terminal homo- and heterosequences of amino acid residues to quantitatively determine nearest neighbor coupling between them with a Ising type model. Results reveal that, depending on the choice of amino acid residue pairs, nearest neighbor interactions either stabilize or destabilize pairs of polyproline II and ß-strand conformations. This leads to a redistribution of population between these conformations and a reduction in conformational entropy. Interactions between residues in polyproline II and turn(helix)-forming conformations seem to be cooperative in most cases, but the respective interaction parameters are subject to large statistical errors.

Do molecular dynamics force fields accurately model Ramachandran distributions of amino acid residues in water?

Molecular dynamics (MD) is a powerful tool for studying intrinsically disordered proteins, however, its reliability depends on the accuracy of the force field. We assess Amber ff19SB, Amber ff14SB, OPLS-AA/M, and CHARMM36m with respect to their capacity to capture intrinsic conformational dynamics of 14 guest residues x (=G, A, L, V, I, F, Y, DP, EP, R, C, N, S, T) in GxG peptides in water. The MD-derived Ramachandran distribution of each guest residue is used to calculate 5 J-coupling constants and amide I' band profiles to facilitate a comparison to spectroscopic data through reduced χ2 functions. We show that the Gaussian model, optimized to best fit the experimental data, outperforms all MD force fields by an order of magnitude. The weaknesses of the MD force fields are: (i) insufficient variability of the polyproline II (pPII) population among the guest residues; (ii) oversampling of antiparallel at the expense of transitional ß-strand region; (iii) inadequate sampling of turn-forming conformations for ionizable and polar residues; and (iv) insufficient guest residue-specificity of the Ramachandran distributions. Whereas Amber ff19SB performs worse than the other three force fields with respect to χ2 values, it accounts for residue-specific pPII content better than the other three force fields. Additional testing of residue-specific RSFF1 and Amber ff14SB combined with TIP4P/2005 on six guest residues x (=A, I, F, DP, R, S) reveals that residue specificity derived from protein coil libraries or an improved water model alone do not result in significantly lower χ2 values.

Repeating Aspartic Acid Residues Prefer Turn-like Conformations in the Unfolded State: Implications for Early Protein Folding.

Protein folding can be described as a motion of the polypeptide chain in a potential energy funnel, where the conformational manifold is narrowed as the chain traverses from a completely unfolded state until it reaches the folded (native) state. The initial folding stages set the tone for this process by substantially narrowing the manifold of accessible conformations. In an ideally unfolded state with no long-range stabilizing forces, local conformations (i.e., residual structures) are likely to drive the folding process. While most amino acid residues tend to predominantly adopt extended structures in unfolded proteins and peptides, aspartic acid exhibits a relatively high intrinsic preference for turn-forming conformations. Regions in an unfolded polypeptide or protein that are rich in aspartic acid residues may therefore be crucial sites for protein folding steps. By combining NMR and vibrational spectroscopies, we observed that the conformational sampling of multiple sequentially neighbored aspartic acid residues in the model peptides GDDG and GDDDG even show an on average higher propensity for turn-forming structures than the intrinsic reference system D in GDG, which suggests that nearest neighbor interactions between adjacent aspartic acid residues stabilize local turn-forming structures. In the presence of the unlike neighbor phenylalanine, nearest neighbor interactions are of a totally different nature in that it they decrease the turn-forming propensities and mutually increase the sampling of polyproline II (pPII) conformations. We hypothesize the structural role of aspartic residues in intrinsically disordered proteins in general, and particularly in small linear motifs, that are very much determined by their respective neighbors.

Concentration Dependence of a Hydrogel Phase Formed by the Deprotonation of the Imidazole Side Chain of Glycylhistidylglycine.

Upon deprotonation of its imidazole group at ∼pH 6, the unblocked tripeptide glycylhistidylglycine (GHG) self-assembles into very long crystalline fibrils on a 10-1000 µm scale which are capable of forming a volume spanning network, that is, hydrogel. The critical peptide concentration for self-assembly at a pH of 6 lies between 50 and 60 mM. The fraction of peptides that self-assemble into fibrils depends on the concentration of deprotonated GHG. While IR spectra seem to indicate the formation of fibrils with standard amyloid fibril ß-sheet structures, vibrational circular dichroism spectra show a strongly enhanced amide I' signal, suggesting that the formed fibrils exhibit significant chirality. The fibril chirality appears to be a function of peptide concentration. Rheological measurements reveal that the rate of gelation is concentration-dependent and that there is an optimum gel strength at intermediate peptide concentrations of ca. 175 mM. This paper outlines the unique properties of the GHG gel phase which is underlain by a surprisingly dense fibril network with an exceptionally strong modulus that make them potential additives for biomedical applications.

Short Peptides as Tunable, Switchable, and Strong Gelators.

This Perspective outlines our current understanding of molecular gels composed of short and ultrashort peptides over the past 20 years. We discuss in detail the state of the art regarding self-assembly mechanisms, structure, thermal stability, and kinetics of fibril and/or network formation. Emphasis is put on the importance of the combined use of spectroscopy and rheology for characterizing and validating self-assembly models. While a range of peptide chemistries are reviewed, we focus our discussion on a unique new class of ultrashort peptide gelators, denoted GxG peptides (x: guest residue), which are capable of forming self-assembled fibril networks. The storage moduli of GxG gels are tunable up to 100 kPa depending on concentration, pH, and/or cosolvent. The sheet structures of the fibrils differ from canonical ß-sheets. When appropriate, each section highlights opportunities for additional research and technologies that would further our understanding.

The impact of thermal history on the structure of glycylalanylglycine ethanol/water gels.

This work revisits several open questions regarding the mechanisms of GAG fibril formation and structure as a function of temperature. The authors recently hypothesized that there is a solubility limit of GAG in ethanol/water that induces self-assembly. In other words, not all peptides can participate in fibrillization and some fraction is still soluble in solution. We show via FTIR spectroscopy that, indeed, free peptides are still present in solution after fibril formation, strongly supporting the solubility model. Furthermore, previous work showed GAG self-assembled into right-handed (phase I) or left-handed (phase II) chiral structures depending on temperature. In this study, we analyze the crystalline structure of phase I and II gels via WAXS and SAXS to compare their crystalline structures and order. Rheological measurements were used to investigate the response of the fibrillar network to temperature. They reveal that the ability of the peptide to self-assemble depends on the solubility at a given temperature and not on thermal history. Furthermore, the gel softening point, the linear viscoelastic gel microstructure, and relaxation spectrum are very similar between phase I and phase II. Overall, the temperature only affects the chirality of the fibrils and the formation kinetics.

Short peptides as predictors for the structure of polyarginine sequences in disordered proteins.

Intrinsically disordered proteins and intrinsically disordered regions are frequently enriched in charged amino acids. Intrinsically disordered regions are regularly involved in important biological processes in which one or more charged residues is the driving force behind a protein-biomolecule interaction. Several lines of experimental and computational evidence suggest that polypeptides and proteins that carry high net charges have a high preference for extended conformations with average end-to-end distances exceeding expectations for self-avoiding random coils. Here, we show that charged arginine residues even in short glycine-capped model peptides (GRRG and GRRRG) significantly affect the conformational propensities of each other when compared with the intrinsic propensities of a mostly unperturbed arginine in the tripeptide GRG. A conformational analysis based on experimentally determined J-coupling constants from heteronuclear NMR spectroscopy and amide I' band profiles from vibrational spectroscopy reveals that nearest-neighbor interactions stabilize extended ß-strand conformations at the expense of polyproline II and turn conformations. The results from molecular dynamics simulations with a CHARMM36m force field and TIP3P water reproduce our results only to a limited extent. The use of the Ramachandran distribution of the central residue of GRRRG in a calculation of end-to-end distances of polyarginines of different length yielded the expected power law behavior. The scaling coefficient of 0.66 suggests that such peptides would be more extended than predicted by a self-avoiding random walk. Our findings thus support in principle theoretical predictions.

Intrinsic Conformational Dynamics of Alanine in Water/Ethanol Mixtures: An Experiment-Driven Molecular Dynamics Study.

In vitro, cationic glycylalanylglycine (GAG) forms a hydrogel in binary mixtures of water and ethanol. In water, alanine residue is known for its high polyproline II (pPII) content. Spectroscopic data, including three J-coupling constants and amide I' profiles, indicate that addition of 42% ethanol to water significantly reduces the pPII content of alanine residue in GAG. Here, experiment-based Gaussian Ramachandran distributions of alanine in GAG at different ethanol fractions are examined and three MD force fields are evaluated with respect to their ability to capture these ethanol-induced conformational changes. MD simulations on monomeric GAG in eight different water/ethanol mixtures within Amber ff14SB, OPLS-AA/M, and CHARMM36m reveal that only Amber ff14SB partially captures the ethanol-induced conformational changes of alanine residue in monomeric GAG when 42% ethanol is added to water. MD simulations of 200 mM GAG ensembles in pure water and in the aqueous solution with 42% ethanol showcase the ability of CHARMM36m to capture the effect of ethanol on the average pPII content of alanine in GAG and provide a plausible explanation for this effect, which may stem from an increased propensity of GAG to form oligomers in the presence of ethanol.

Formation of peptide-based oligomers in dimethylsulfoxide: identifying the precursor of fibril formation.

The well-studied dipeptide fluorenylmethyloxycarbonyl-di-phenylalanine (FmocFF) forms a rigid hydrogel upon dissolving in dimethylsulfoxide (DMSO) and dilution in H2O. Here, we explored the pre-aggregation of the peptide in pure DMSO by vibrational spectroscopies, X-ray powder diffraction and dynamic light scattering. Our results show an equilibrium between a dominant population of amorphous oligomers (on a length scale of 2 nm) and a small number of protofibrils/fibrils (on a length scale of 30 nm in the centimolar and of 200 nm in the sub-molar region). To probe the mechanism underlying the formation of these protofilaments, we measured the 1H-NMR, IR and visible Raman spectra of DMSO containing different FmocFF concentrations, ranging between 10 and 300 mM. Our data reveal that interpeptide hydrogen bonding leads to the self-assembly of FmocFF in the centimolar region, while π-π stacking between Fmoc-groups is observed above 100 mM. The high 3J(HNHCα) coupling constant of the N-terminal amide proton indicates that the Fmoc end-cap of the peptide locks the N-terminal residue into a conformational ensemble centered at a φ-value of ca. -120°, which corresponds to a parallel ß-sheet type conformation. The 3J(HNHCα) coupling constant of the C-terminal residue is indicative of a polyproline II (pPII)/ßt mixture. Our results suggest that the gelation of FmocFF caused by the addition of a small amount of water to DMSO mixtures is facilitated by the formation of disordered protofibrils in pure DMSO.

Glycine in Water Favors the Polyproline II State.

Conformational preferences of amino acid residues in water are determined by the backbone and side-chain properties. Alanine is known for its high polyproline II (pPII) propensity. The question of relative contributions of the backbone and side chain to the conformational preferences of alanine and other amino acid residues in water is not fully resolved. Because glycine lacks a heavy-atom side chain, glycine-based peptides can be used to examine to which extent the backbone properties affect the conformational space. Here, we use published spectroscopic data for the central glycine residue of cationic triglycine in water to demonstrate that its conformational space is dominated by the pPII state. We assess three commonly used molecular dynamics (MD) force fields with respect to their ability to capture the conformational preferences of the central glycine residue in triglycine. We show that pPII is the mesostate that enables the functional backbone groups of the central residue to form the most hydrogen bonds with water. Our results indicate that the pPII propensity of the central glycine in GGG is comparable to that of alanine in GAG, implying that the water-backbone hydrogen bonding is responsible for the high pPII content of these residues.