Folding simulations of Trp‐cage mini protein in explicit solvent using biasing potential replica‐exchange molecular dynamics simulations

Replica exchange molecular dynamics (RexMD) simulations are frequently used for studying structure formation and dynamics of peptides and proteins. A significant drawback of standard temperature RexMD is, however, the rapid increase of the replica number with increasing system size to cover a desired temperature range. A recently developed Hamiltonian RexMD method has been used to study folding of the Trp‐cage protein. It employs a biasing potential that lowers the backbone dihedral barriers and promotes peptide backbone transitions along the replica coordinate. In two independent applications of the biasing potential RexMD method including explicit solvent and starting from a completely unfolded structure the formation of near‐native conformations was observed after 30–40 ns simulation time. The conformation representing the most populated cluster at the final simulation stage had a backbone root mean square deviation of ～1.3 Å from the experimental structure. This was achieved with a very modest number of five replicas making it well suited for peptide and protein folding and refinement studies including explicit solvent. In contrast, during five independent continuous 70 ns molecular dynamics simulations formation of collapsed states but no near native structure formation was observed. The simulations predict a largely collapsed state with a significant helical propensity for the helical domain of the Trp‐cage protein already in the unfolded state. Hydrogen bonded bridging water molecules were identified that could play an active role by stabilizing the arrangement of the helical domain with respect to the rest of the chain already in intermediate states of the protein. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Folding mechanism of three structurally similar β-sheet proteins

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly β-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, ΔG) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences. Proteins 33:107–118, 1998. © 1998 Wiley-Liss, Inc.     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Oligomerization of a symmetric β‐trefoil protein in response to folding nucleus perturbation

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl‐terminal region of the (internally located) FN of the de novo designed purely‐symmetric β‐trefoil protein Symfoil‐4P. This mutation involves wholesale replacement of a repeating trefoil‐fold motif with a “blade” motif from a β‐propeller protein, and postulated to trap that region of the Symfoil‐4P FN in a nonproductive folding intermediate. The resulting protein (termed “Bladefoil”) is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.     

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.     

All-atom folding of the three-helix HIV accessory protein with an adaptive parallel tempering method

All-atom protein structure prediction from the amino acid sequence alone remains an important goal of biophysical chemistry. Recent progress in force field development and validation suggests that the PFF01 free-energy force field correctly predicts the native conformation of various helical proteins as the global optimum of its free-energy surface. Reproducible protein structure prediction requires the availability of efficient optimization methods to locate the global minima of such complex potentials. Here we investigate an adapted version of the parallel tempering method as an efficient parallel stochastic optimization method for protein structure prediction. Using this approach we report the reproducible all-atom folding of the three-helix 40 amino acid HIV accessory protein from random conformations to within 2.4 A backbone RMS deviation from the experimental structure with modest computational resources.     

Monte carlo simulations of protein folding. II. Application to protein A,ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338–352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 Å from the native Cα trace, and the best coordinate rms from crambin is 3.18 Å. For ROP monomer, the lowest coordinate rms from equivalent Cαs of ROP dimer is 3.65 Å. Thus, for two simple helical proteins and a small α/β protein, the ability to predict protein structure from sequence has been demonstrated. © 1994 John Wiley & Sons, Inc.     

Benchmarking of TASSER in the ab initio limit

A significant number of protein sequences in a given proteome have no obvious evolutionarily related protein in the database of solved protein structures, the PDB. Under these conditions, ab initio or template-free modeling methods are the sole means of predicting protein structure. To assess its expected performance on proteomes, the TASSER structure prediction algorithm is benchmarked in the ab initio limit on a representative set of 1129 nonhomologous sequences ranging from 40 to 200 residues that cover the PDB at 30% sequence identity and which adopt alpha, alpha + beta, and beta secondary structures. For sequences in the 40-100 (100-200) residue range, as assessed by their root mean square deviation from native, RMSD, the best of the top five ranked models of TASSER has a global fold that is significantly close to the native structure for 25% (16%) of the sequences, and with a correct identification of the structure of the protein core for 59% (36%). In the absence of a native structure, the structural similarity among the top five ranked models is a moderately reliable predictor of folding accuracy. If we classify the sequences according to their secondary structure content, then 64% (36%) of alpha, 43% (24%) of alpha + beta, and 20% (12%) of beta sequences in the 40-100 (100-200) residue range have a significant TM-score (TM-score > or = 0.4). TASSER performs best on helical proteins because there are less secondary structural elements to arrange in a helical protein than in a beta protein of equal length, since the average length of a helix is longer than that of a strand. In addition, helical proteins have shorter loops and dangling tails. If we exclude these flexible fragments, then TASSER has similar accuracy for sequences containing the same number of secondary structural elements, irrespective of whether they are helices and/or strands. Thus, it is the effective configurational entropy of the protein that dictates the average likelihood of correctly arranging the secondary structure elements.     

Hierarchical protein folding pathways: a computational study of protein fragments

We have previously presented a building block folding model. The model postulates that protein folding is a hierarchical top-down process. The basic unit from which a fold is constructed, referred to as a hydrophobic folding unit, is the outcome of combinatorial assembly of a set of "building blocks." Results obtained by the computational cutting procedure yield fragments that are in agreement with those obtained experimentally by limited proteolysis. Here we show that as expected, proteins from the same family give very similar building blocks. However, different proteins can also give building blocks that are similar in structure. In such cases the building blocks differ in sequence, stability, contacts with other building blocks, and in their 3D locations in the protein structure. This result, which we have repeatedly observed in many cases, leads us to conclude that while a building block is influenced by its environment, nevertheless, it can be viewed as a stand-alone unit. For small-sized building blocks existing in multiple conformations, interactions with sister building blocks in the protein will increase the population time of the native conformer. With this conclusion in hand, it is possible to develop an algorithm that predicts the building block assignment of a protein sequence whose structure is unknown. Toward this goal, we have created sequentially nonredundant databases of building block sequences. A protein sequence can be aligned against these, in order to be matched to a set of potential building blocks.     

Sub-microsecond protein folding

We have investigated the structure, equilibria, and folding kinetics of an engineered 35-residue subdomain of the chicken villin headpiece, an ultrafast-folding protein. Substitution of two buried lysine residues by norleucine residues stabilizes the protein by 1 kcal/mol and increases the folding rate sixfold, as measured by nanosecond laser T-jump. The folding rate at 300 K is (0.7 micros)(-1) with little or no temperature dependence, making this protein the first sub-microsecond folder, with a rate only twofold slower than the theoretically predicted speed limit. Using the 70 ns process to obtain the effective diffusion coefficient, the free energy barrier height is estimated from Kramers theory to be less than approximately 1 kcal/mol. X-ray crystallographic determination at 1A resolution shows no significant change in structure compared to the single-norleucine-substituted molecule and suggests that the increased stability is electrostatic in origin. The ultrafast folding rate, very accurate X-ray structure, and small size make this engineered villin subdomain an ideal system for simulation by atomistic molecular dynamics with explicit solvent.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Physical principles of protein structure and protein folding

Physical principles determining the protein structure and protein folding are reviewed: (i) the molecular theory of protein secondary structure and the method of its prediction based on this theory; (ii) the existence of a limited set of thermodynamically favourable folding patterns of α- and β-regions in a compact globule which does not depend on the details of the amino acid sequence; (iii) the moderns approaches to the prediction of the folding patterns of α- and β-regions in concrete proteins; (iv) experimental approaches to the mechanism of protein folding. The review reflects theoretical and experimental works of the author and his collaborators as well as those of other groups.     

Ab initio folding of a trefoil‐fold motif reveals structural similarity with a β‐propeller blade motif

Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain‐swapped arrangement at the interface of the N‐ and C‐termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N‐ and C‐termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β‐trefoil protein fold is a threefold‐symmetric architecture where the repeating ~42‐mer “trefoil‐fold” motif assembles via a domain‐swapped arrangement. The trefoil‐fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β‐trefoil trimeric assembly. The trefoil‐fold sequence is not predicted to adopt the trefoil‐fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact “blade” motif from the β‐propeller architecture. Expression of a trefoil‐fold sequence and circular permutants shows that only the wild‐type N‐terminal motif definition yields an intact β‐trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil‐fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil‐fold structural features, but is more structurally homologous to a β‐propeller blade motif.     

Transformation of an alpha-helix peptide into a beta-hairpin induced by addition of a fragment results in creation of a coexisting state

Intrinsic rules of determining the tertiary structure of a protein have been unknown partly because physicochemical factors that contribute to stabilization of a protein structure cannot be represented as a linear combination of local interactions. To clarify the rules on the nonlinear term caused by nonlocal interaction in a protein, we tried to transform a peptide that has a fully helical structure (Target Peptide or TP) into a peptide that has a beta-hairpin structure (Designed Peptide or DP) by adding seven residues to the C terminus of TP. According to analyses of nuclear magnetic resonance measurements, while the beta-hairpin structure is stabilized in some DPs, it is evident that the helical structure observed in TP is also persistent and even extended throughout the length of the molecule. As a result, we have produced a peptide molecule that contains both the alpha-helix and beta-hairpin conformation at an almost equally populated level. The helical structures contained in these DPs were more stable than the helix in TP, suggesting that stabilizing one conformation does not result in destabilizing the other conformation. These DPs can thus be regarded as an isolated peptide version of the chameleon sequence, which has the capability of changing the secondary structure depending on the context of the surrounding environment in a protein structure. The fact that the transformation of one secondary structure caused stabilization of both the original and the induced structure would shed light on the mechanism of protein folding.     

Design and characterization of a model αβ peptide

CD and nmr spectroscopy were used to compare the conformational properties of two related peptides. One of the peptides, Model AB, was designed to adopt a helix-turn-extended strand (αβ) tertiary structure in water that might be stabilized by hydrophobic interactions between two leucine residues in the amino-terminal segment and two methionine residues in the carboxyl terminal segment. The other peptide, AB Helix, has the same amino acid sequence as Model AB except that it lacks the-Pro-Met-Thr-Met-Thr-Gly segment at the carboxyl-terminus. Although the carboxyl-terminal segment of Model AB was found to be unstructured, its presence increases the number of residues in a helical conformation, shifts the pKas of three ionizable side chains by 1 pH unit or more compared to an unstructured peptide, stabilizes the peptide as a monomer in high concentrations of ammonium sulfate, increases the conformational stability of residues at the terminal ends of the helix, and results in many slowly exchanging amide protons throughout the entire backbone of the peptide. These results suggest that interactions between adjacent segments in a small peptide can have significant structure organizing effects. Similar kinds of interactions may be important in determining the structure of early intermediates in protein folding and may be useful in the de novo design of independently folding peptides. © 1995 John Wiley & Sons, Inc.     

Marginally hydrophobic transmembrane α‐helices shaping membrane protein folding

Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment.     

Reduced alphabet for protein folding prediction

What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28‐letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28‐letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least 10 folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design. Proteins 2015; 83:631–639. © 2015 Wiley Periodicals, Inc.     

Toward an improved structural model of the frog-skin antimicrobial peptide esculentin-1b(1-18)

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptide's folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role.