Comparing the energy landscapes for native folding and aggregation of PrP

Protein sequences are evolved to encode generally one folded structure, out of a nearly infinite array of possible folds. Underlying this code is a funneled free energy landscape that guides folding to the native conformation. Protein misfolding and aggregation are also a manifestation of free-energy landscapes. The detailed mechanisms of these processes are poorly understood, but often involve rare, transient species and a variety of different pathways. The inherent complexity of misfolding has hampered efforts to measure aggregation pathways and the underlying energy landscape, especially using traditional methods where ensemble averaging obscures important rare and transient events. We recently studied the misfolding and aggregation of prion protein by examining 2 monomers tethered in close proximity as a dimer, showing how the steps leading to the formation of a stable aggregated state can be resolved in the single-molecule limit and the underlying energy landscape thereby reconstructed. This approach allows a more quantitative comparison of native folding versus misfolding, including fundamental differences in the dynamics for misfolding. By identifying key steps and interactions leading to misfolding, it should help to identify potential drug targets. Here we describe the importance of characterizing free-energy landscapes for aggregation and the challenges involved in doing so, and we discuss how single-molecule studies can help test proposed structural models for PrP aggregates.     

Parser for protein folding units

General patterns of protein structural organization have emerged from studies of hundreds of structures elucidated by X-ray crystallography and nuclear magnetic resonance. Structural units are commonly identified by visual inspection of molecular models using qualitative criteria. Here, we propose an algorithm for identification of structural units by objective, quantitative criteria based on atomic interactions. The underlying physical concept is maximal interactions within each unit and minimal interaction between units (domains). In a simple harmonic approximation, interdomain dynamics is determined by the strength of the interface and the distribution of masses. The most likely domain decomposition involves units with the most correlated motion, or largest interdomain fluctuation time. The decomposition of a convoluted 3-D structure is complicated by the possibility that the chain can cross over several times between units. Grouping the residues by solving an eigenvalue problem for the contact matrix reduces the problem to a one-dimensional search for all reasonable trial bisections. Recursive bisection yields a tree of putative folding units. Simple physical criteria are used to identify units that could exist by themselves. The units so defined closely correspond to crystallographers' notion of structural domains. The results are useful for the analysis of folding principles, for modular protein design and for protein engineering. © 1994 Wiley-Liss, Inc.     

对称蛋白质序列与结构关系研究

进化的观点认为,蛋白质结构的对称性是基因复制和融合的结果,但是由于在长期进化过程中的氨基酸突变,绝大多数现有的蛋白质序列都失去了这种直观的重复性特征。该文简要地回顾了国际上发展的寻找蛋白质序列中重复片段的方法,重点介绍了作者自己提出的分析蛋白质序列和结构对称性的方法以及在蛋白质对称结构形成机理方面的初步工作,并系统分析了各类对称折叠子的序列与结构关系,发现它们的序列都具有隐含的与结构相同的对称性,或者说序列的对称性决定结构的对称性。     

Increased sequence hydrophobicity reduces conformational specificity: A mutational case study of the Arc repressor protein

The amino-acid sequences of soluble, globular proteins must have hydrophobic residues to form a stable core, but excess sequence hydrophobicity can lead to loss of native state conformational specificity and aggregation. Previous studies of polar-to-hydrophobic mutations in the β-sheet of the Arc repressor dimer showed that a single substitution at position 11 (N11L) leads to population of an alternate dimeric fold in which the β-sheet is replaced by helix. Two additional hydrophobic mutations at positions 9 and 13 (Q9V and R13V) lead to population of a differently folded octamer along with both dimeric folds. Here we conduct a comprehensive study of the sequence determinants of this progressive loss of fold specificity. We find that the alternate dimer-fold specifically results from the N11L substitution and is not promoted by other hydrophobic substitutions in the β-sheet. We also find that three highly hydrophobic substitutions at positions 9, 11, and 13 are necessary and sufficient for oligomer formation, but the oligomer size depends on the identity of the hydrophobic residue in question. The hydrophobic substitutions increase thermal stability, illustrating how increased hydrophobicity can increase folding stability even as it degrades conformational specificity. The oligomeric variants are predicted to be aggregation-prone but may be hindered from doing so by proline residues that flank the β-sheet region. Loss of conformational specificity due to increased hydrophobicity can manifest itself at any level of structure, depending upon the specific mutations and the context in which they occur.     

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.     

CHOP proteins into structural domain-like fragments

We developed a method CHOP dissecting proteins into domain-like fragments. The basic idea was to cut proteins beginning from very reliable experimental information (PDB), proceeding to expert annotations of domain-like regions (Pfam-A), and completing through cuts based on termini of known proteins. In this way, CHOP dissected more than two thirds of all proteins from 62 proteomes. Analysis of our structural domain-like fragments revealed four surprising results. First, >70% of all dissected proteins contained more than one fragment. Second, most domains spanned on average over approximately 100 residues. This average was similar for eukaryotic and prokaryotic proteins, and it is also valid-although previously not described-for all proteins in the PDB. Third, single-domain proteins were significant longer than most domains in multidomain proteins. Fourth, three fourths of all domains appeared shorter than 210 residues. We believe that our CHOP fragments constituted an important resource for functional and structural genomics. Nevertheless, our main motivation to develop CHOP was that the single-linkage clustering method failed to adequately group full-length proteins. In contrast, CLUP-the simple clustering scheme CLUP introduced here-succeeded largely to group the CHOP fragments from 62 proteomes such that all members of one cluster shared a basic structural core. CLUP found >63,000 multi- and >118,000 single-member clusters. Although most fragments were restricted to a particular cluster, approximately 24% of the fragments were duplicated in at least two clusters. Our thresholds for grouping two fragments into the same cluster were rather conservative. Nevertheless, our results suggested that structural genomics initiatives have to target >30,000 fragments to at least cover the multimember clusters in 62 proteomes.     

Amino acid sequence predicts folding rate for middle-size two-state proteins

The significant correlation between protein folding rates and the sequence-predicted secondary structure suggests that folding rates are largely determined by the amino acid sequence. Here, we present a method for predicting the folding rates of proteins from sequences using the intrinsic properties of amino acids, which does not require any information on secondary structure prediction and structural topology. The contribution of residue to the folding rate is expressed by the residue's Omega value. For a given residue, its Omega depends on the amino acid properties (amino acid rigidity and dislike of amino acid for secondary structures). Our investigation achieves 82% correlation with folding rates determined experimentally for simple, two-state proteins studied until the present, suggesting that the amino acid sequence of a protein is an important determinant of the protein-folding rate and mechanism.     

Knowledge-based model building of proteins: concepts and examples.

We describe how to build protein models from structural templates. Methods to identify structural similarities between proteins in cases of significant, moderate to low, or virtually absent sequence similarity are discussed. The detection and evaluation of structural relationships is emphasized as a central aspect of protein modeling, distinct from the more technical aspects of model building. Computational techniques to generate and complement comparative protein models are also reviewed. Two examples, P-selectin and gp39, are presented to illustrate the derivation of protein model structures and their use in experimental studies.     

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.     

Computer modeling and folding of four-helix bundles

In the context of simplified models of globular proteins, the requirements for the unique folding to a four-helix bundle have been addressed through a new Monte Carlo procedure. In particular, the relative importance of secondary versus tertiary interactions in determining the nature of the folded structure is examined. Various cases spanning the extremes where tertiary interactions completely dominate to that where tertiary interactions are negligible have been explored. Not surprisingly, the folding to unique four-helix bundles is found to depend on an adequate balance of the secondary and tertiary interactions. Moreover, because the simplified model is composed of spheres representing α-carbons and side chains, the geometry of the latter being based on small real amino acids, the role played by the side chains, and the problems associated with packing and hard-core repulsions, are considered. Also, possible folding intermediates and their relationship with the experimentally observed molten globule state are explored. From these studies, a general set of rules is extracted which should aid in the further design of more detailed protein models adequate to more fully investigate the protein folding problem. Finally, the relationship between our conclusions and experimental work with specifically designed sequences is briefly discussed. © 1993 Wiley-Liss, Inc.     

Optimization of the electrostatic interactions in proteins of different functional and folding type

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α-helical or β-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.     

Conformational analysis of polypeptides and proteins for the study of protein folding, molecular recognition, and molecular design

Conformational energy calculations provide an understanding as to how interatomic interactions lead to the three-dimensional structures of polypeptides and proteins, and how these molecules interact with other molecules. Illustrative results of such calculations pertain to model systems (-helices and -sheets, and interactions between them), to various open-chain and cyclic peptides, to fibrous proteins, to globular proteins, and to enzyme-substrate complexes. In most cases, the validity of the computations is established by experimental tests of the predicted structures.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985. This paper first appeared in the Israel Journal of Chemistry, Vol. 27, 1986.     

A protein sequence fitness function for identifying natural and nonnatural proteins

The infinitesimally small sequence space naturally scouted in the millions of years of evolution suggests that the natural proteins are constrained by some functional prerequisites and should differ from randomly generated sequences. We have developed a protein sequence fitness scoring function that implements sequence and corresponding secondary structural information at tripeptide levels to differentiate natural and nonnatural proteins. The proposed fitness function is extensively validated on a dataset of about 210 000 natural and nonnatural protein sequences and benchmarked with existing methods for differentiating natural and nonnatural proteins. The high sensitivity, specificity, and percentage accuracy (0.81%, 0.95%, and 91% respectively) of the fitness function demonstrates its potential application for sampling the protein sequences with higher probability of mimicking natural proteins. Moreover, the four major classes of proteins (α proteins, β proteins, α/β proteins, and α + β proteins) are separately analyzed and β proteins are found to score slightly lower as compared to other classes. Further, an analysis of about 250 designed proteins (adopted from previously reported cases) helped to define the boundaries for sampling the ideal protein sequences. The protein sequence characterization aided by the proposed fitness function could facilitate the exploration of new perspectives in the design of novel functional proteins.     

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.     

Comparing side chain packing in soluble proteins,protein‐protein interfaces,and transmembrane proteins

We compare side chain prediction and packing of core and non‐core regions of soluble proteins, protein‐protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high‐resolution crystal structures of these 3 protein classes. We show that the solvent‐inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein‐protein interfaces and in the membrane‐exposed regions of transmembrane proteins can be predicted by the hard‐sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent‐inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within ) up to a relative solvent accessibility, , for all 3 protein classes. Our results show that % of the interface regions in protein complexes are “core”, that is, densely packed with side chain conformations that can be accurately predicted using the hard‐sphere model. We propose packing fraction as a metric that can be used to distinguish real protein‐protein interactions from designed, non‐binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Designing repeat proteins: well-expressed,soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.     

Folding of multidomain proteins: Biophysical consequences of tethering even in apparently independent folding

Most eukaryotic and a substantial fraction of prokaryotic proteins are composed of more than one domain. The tethering of these evolutionary, structural, and functional units raises, among others, questions regarding the folding process of conjugated domains. Studying the folding of multidomain proteins in silico enables one to identify and isolate the tethering‐induced biophysical determinants that govern crosstalks generated between neighboring domains. For this purpose, we carried out coarse‐grained and atomistic molecular dynamics simulations of two two‐domain constructs from the immunoglobulin‐like β‐sandwich fold. Each of these was experimentally shown to behave as the “sum of its parts,” that is, the thermodynamic and kinetic folding behavior of the constituent domains of these constructs seems to occur independently, with the folding of each domain uncoupled from the folding of its partner in the two‐domain construct. We show that the properties of the individual domains can be significantly affected by conjugation to another domain. The tethering may be accompanied by stabilizing as well as destabilizing factors whose magnitude depends on the size of the interface, the length, and the flexibility of the linker, and the relative stability of the domains. Accordingly, the folding of a multidomain protein should not be viewed as the sum of the folding patterns of each of its parts, but rather, it involves abrogating several effects that lead to this outcome. An imbalance between these effects may result in either stabilization or destabilization owing to the tethering. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Intermediates and the folding of proteins L and G

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted beta-1 and beta-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third beta-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first-order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.