Exploring the free energy landscape of a model β‐hairpin peptide and its isoform

Secondary structural transitions from α‐helix to β‐sheet conformations are observed in several misfolding diseases including Alzheimer's and Parkinson's. Determining factors contributing favorably to the formation of each of these secondary structures is therefore essential to better understand these disease states. β‐hairpin peptides form basic components of anti‐parallel β‐sheets and are suitable model systems for characterizing the fundamental forces stabilizing β‐sheets in fibrillar structures. In this study, we explore the free energy landscape of the model β‐hairpin peptide GB1 and its E2 isoform that preferentially adopts α‐helical conformations at ambient conditions. Umbrella sampling simulations using all‐atom models and explicit solvent are performed over a large range of end‐to‐end distances. Our results show the strong preference of GB1 and the E2 isoform for β‐hairpin and α‐helical conformations, respectively, consistent with previous studies. We show that the unfolded states of GB1 are largely populated by misfolded β‐hairpin structures which differ from each other in the position of the β‐turn. We discuss the energetic factors contributing favorably to the formation of α‐helix and β‐hairpin conformations in these peptides and highlight the energetic role of hydrogen bonds and non‐bonded interactions. Proteins 2014; 82:2394–2402. © 2014 Wiley Periodicals, Inc.     

Osmolyte perturbation reveals conformational equilibria in spin‐labeled proteins

Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin‐labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site‐directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native‐like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

High-Resolution Structure of a Protein Spin-Label in a Solvent-Exposed β-Sheet and Comparison with DEER Spectroscopy

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed α-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior β-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the H(α) and S(δ) hydrogen bond that is common to α-helical sites is absent at this interior β-strand residue. Variable temperature continuous wave (CW) experiments of the β-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the H(α) and S(δ) interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 ± 1 ?. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within ～2 ?. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER.     

Helix stability of oligoglycine,oligoalanine, and oligo‐β‐alanine dodecamers reflected by hydrogen‐bond persistence

Helices are important structural/recognition elements in proteins and peptides. Stability and conformational differences between helices composed of α‐ and β‐amino acids as scaffolds for mimicry of helix recognition has become a theme in medicinal chemistry. Furthermore, helices formed by β‐amino acids are experimentally more stable than those formed by α‐amino acids. This is paradoxical because the larger sizes of the hydrogen‐bonding rings required by the extra methylene groups should lead to entropic destabilization. In this study, molecular dynamics simulations using the second‐generation force field, AMOEBA (Ponder, J.W., et al., Current status of the AMOEBA polarizable force field. J Phys Chem B, 2010. 114 (8): p. 2549–64.) explored the stability and hydrogen‐bonding patterns of capped oligo‐β‐alanine, oligoalanine, and oligoglycine dodecamers in water. The MD simulations showed that oligo‐β‐alanine has strong acceptor+2 hydrogen bonds, but surprisingly did not contain a large content of 3₁₂‐helical structures, possibly due to the sparse distribution of the 3₁₂‐helical structure and other structures with acceptor+2 hydrogen bonds. On the other hand, despite its backbone flexibility, the β‐alanine dodecamer had more stable and persistent <3.0 Å hydrogen bonds. Its structure was dominated more by multicentered hydrogen bonds than either oligoglycine or oligoalanine helices. The 3₁ (PII) helical structure, prevalent in oligoglycine and oligoalanine, does not appear to be stable in oligo‐β‐alanine indicating its competition with other structures (stacking structure as indicated by MD analyses). These differences are among the factors that shape helical structural preferences and the relative stabilities of these three oligopeptides. Proteins 2014; 82:3043–3061. © 2014 Wiley Periodicals, Inc.     

Propensities of peptides containing the Asn‐Gly segment to form β‐turn and β‐hairpin structures

The propensities of peptides that contain the Asn‐Gly segment to form β‐turn and β‐hairpin structures were explored using the density functional methods and the implicit solvation model in CH₂Cl₂ and water. The populations of preferred β‐turn structures varied depending on the sequence and solvent polarity. In solution, β‐hairpin structures with βI′ turn motifs were most preferred for the heptapeptides containing the Asn‐Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X‐ray structures. The sequence, H‐bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β‐hairpin structure of each heptapeptide, although the β‐turn segments played a role in promoting the formation of β‐hairpin structures and the β‐hairpin propensity varied. In the heptapeptides containing the Asn‐Gly segment, the β‐hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β‐turns and β‐hairpins containing the Asn‐Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics and the design of binding epitopes for protein–protein and protein–nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653–664, 2016.     

β‐Bulges: Extensive structural analyses of β‐sheets irregularities

β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

Computer modeling of nitroxide spin labels on proteins

Electron paramagnetic resonance using site‐directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron–electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F‐BAR), endophilin, and α‐synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r² = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 35–44, 2012.     

Geometrical principles of homomeric β‐barrels and β‐helices: Application to modeling amyloid protofilaments

Examples of homomeric β‐helices and β‐barrels have recently emerged. Here we generalize the theory for the shear number in β‐barrels to encompass β‐helices and homomeric structures. We introduce the concept of the “β‐strip,” the set of parallel or antiparallel neighboring strands, from which the whole helix can be generated giving it n‐fold rotational symmetry. In this context, the shear number is interpreted as the sum around the helix of the fixed register shift between neighboring identical β‐strips. Using this approach, we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α‐hemolysin, T4 phage spike, cylindrin, and the HET‐s(218‐289) prion. From reported dimensions measured by X‐ray fiber diffraction on amyloid fibrils, the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in‐register β‐strands folded into a “β‐strip helix.” Results suggest both stabilization of an individual β‐strip helix and growth by addition of further β‐strip helices can involve the same pair of sequence segments associating with β‐sheet hydrogen bonding at the same register shift. This process would be aided by a repeat sequence. Hence, understanding how the register shift (as the distance between repeat sequences) relates to helical dimensions will be useful for nanotube design.     

An amino acid code for β‐sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob‐socket model in an investigation of β‐sheet packing. Over a comprehensive set of β‐sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four‐residue packing cliques necessary to describe β‐sheet packing were characterized. Both occur between two adjacent hydrogen bonded β‐strands. First, defining the secondary structure packing within β‐sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β‐sheets, the knob‐socket XY:H+B consists of a three‐residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob‐sockets are found: side‐chain and main‐chain sockets. The amino acid composition of the pockets and knob‐sockets reveal the sequence specificity of β‐sheet packing. For β‐sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side‐chain and main‐chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β‐sheet structure and provide an intuitive topological mapping of β‐sheet packing. Proteins 2014; 82:2128–2140. © 2014 Wiley Periodicals, Inc.     

Local sequence of protein β‐strands influences twist and bend angles

β‐Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat β‐sheets, suggesting that side chains influence β‐structures. We therefore investigated the relationship between amino acid composition and twists or bends of β‐strands. We calculated and statistically analyzed the twist and bend angles of short frames of β‐strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of β‐strands, suggesting that the side chains of these residues likely do not affect β‐strand structure. In contrast, the majority of serine, threonine, and asparagine side‐chains in β‐strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress β‐strand twisting. Proteins 2014; 82:1484–1493. © 2014 Wiley Periodicals, Inc.     

A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational and aggregation properties of the 1–93 fragment of apolipoprotein A‐I

Several disease‐linked mutations of apolipoprotein A‐I, the major protein in high‐density lipoprotein (HDL), are known to be amyloidogenic, and the fibrils often contain N‐terminal fragments of the protein. Here, we present a combined computational and experimental study of the fibril‐associated disordered 1–93 fragment of this protein, in wild‐type and mutated (G26R, S36A, K40L, W50R) forms. In atomic‐level Monte Carlo simulations of the free monomer, validated by circular dichroism spectroscopy, we observe changes in the position‐dependent β‐strand probability induced by mutations. We find that these conformational shifts match well with the effects of these mutations in thioflavin T fluorescence and transmission electron microscopy experiments. Together, our results point to molecular mechanisms that may have a key role in disease‐linked aggregation of apolipoprotein A‐I.     

An alternative structural isoform in amyloid‐like aggregates formed from thermally denatured human γD‐crystallin

The eye lens protein γD‐crystallin contributes to cataract formation in the lens. In vitro experiments show that γD‐crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV‐B photodamage results in its C‐terminal domain forming the β‐sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet‐like aggregates that contain cross‐linked oligomers of the protein, according to transmission electron microscopy and SDS‐PAGE. We use two‐dimensional infrared spectroscopy to show that these aggregates have an amyloid‐like secondary structure with extended β‐sheets, and use isotope dilution experiments to show that each protein contributes approximately one β‐strand to each β‐sheet in the aggregates. Using segmental ¹³C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N‐terminal and C‐terminal domains contribute to β‐sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N‐terminal domain into the fiber structure to intermolecular cross linking.     

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔG_U = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational analysis of the full‐length M2 protein of the influenza A virus using solid‐state NMR

The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N‐terminal ectodomain and the C‐terminal cytoplasmic tail remains largely unknown. Using two‐dimensional (2D) magic‐angle‐spinning solid‐state NMR, we have investigated the secondary structure and dynamics of full‐length M2 (M2FL) and found them to depend on the membrane composition. In 2D ¹³C DARR correlation spectra, 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)‐bound M2FL exhibits several peaks at β‐sheet chemical shifts, which result from water‐exposed extramembrane residues. In contrast, M2FL bound to cholesterol‐containing membranes gives predominantly α‐helical chemical shifts. Two‐dimensional J‐INADEQUATE spectra and variable‐temperature ¹³C spectra indicate that DMPC‐bound M2FL is highly dynamic while the cholesterol‐containing membranes significantly immobilize the protein at physiological temperature. Chemical‐shift prediction for various secondary‐structure models suggests that the β‐strand is located at the N‐terminus of the DMPC‐bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain‐truncated M2(21–97), which no longer exhibits β‐sheet chemical shifts in the DMPC‐bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol‐rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β‐strand location suggests that chemical‐shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.     

Structural motifs in which β‐strands are clipped together with the П‐like module

In this study, the structural motifs that can be represented as combinations of small motifs such as β‐hairpins, S‐, and Z‐like β‐sheets and βαβ‐units, and the П‐like module are described and analyzed. The П‐module consists of connected elements of the β‐strand‐loop‐β‐strand type arranged in space so that its overall fold resembles a clip or the Greek letter П. In proteins, the П‐module itself and the structural motifs containing it exhibit unique overall folds and have specific sequence patterns of the key hydrophobic, hydrophilic and glycine residues. All this together enables us to conclude that these structural motifs can fold independently of the remaining part of the molecule and can act as nuclei and/or “ready‐made” building blocks in protein folding.     

Determination of the secondary structural elements of chicken liver fatty acid binding protein by two‐dimensional homonuclear NMR

A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence‐specific ¹H resonance assignment was achieved from homonuclear two‐dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel β‐strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the β‐sheet conformation between the first and the last β‐strand in the protein sequence, as well as a helical loop at the N‐terminus of the polypeptide chain. © 1999 John Wiley & Sons, Inc. Biopoly 50: 1–11, 1999