Universally conserved positions in protein folds: reading evolutionary signals about stability, folding kinetics and function.

Here, we provide an analysis of molecular evolution of five of the most populated protein folds: immunoglobulin fold, oligonucleotide-binding fold, Rossman fold, alpha/beta plait, and TIM barrels. In order to distinguish between "historic", functional and structural reasons for amino acid conservations, we consider proteins that acquire the same fold and have no evident sequence homology. For each fold we identify positions that are conserved within each individual family and coincide when non-homologous proteins are structurally superimposed. As a baseline for statistical assessment we use the conservatism expected based on the solvent accessibility. The analysis is based on a new concept of "conservatism-of-conservatism". This approach allows us to identify the structural features that are stabilized in all proteins having a given fold, despite the fact that actual interactions that provide such stabilization may vary from protein to protein. Comparison with experimental data on thermodynamics, folding kinetics and function of the proteins reveals that such universally conserved clusters correspond to either: (i) super-sites (common location of active site in proteins having common tertiary structures but not function) or (ii) folding nuclei whose stability is an important determinant of folding rate, or both (in the case of Rossman fold). The analysis also helps to clarify the relation between folding and function that is apparent for some folds.     

Construction and characterization of chimeric proteins composed of type-1 and type-2 periplasmic binding proteins MglB and ArgT

The respective type-1 and type-2 periplasmic binding proteins (PBPs) MglB and ArgT are believed to have evolved from a common ancestor into siblings showing topological differences in their main chain connectivity. At first glance, they show similar structure. But, more detailed examination reveals that the chain connectivity of ArgT is more convoluted than that of MglB. Reflecting that complexity, the folding of ArgT is complicated and involves intermediate folds. On the other hand, the folding of MglB is a simple two-state transition. In the present study, we constructed and characterized several chimeras made up of various subdomains of MglB and ArgT with the aim of gaining insight into the evolution of protein folding and protein structure. Although these chimeras did not fold as compactly as their parental proteins, some did exhibit cooperative folding, which suggests that novel proteins with new connectivity and new folding pathways could have emerged at a fairly high rate throughout the evolution of proteins.     

Vertical decomposition with Genetic Algorithm for Multiple Sequence Alignment

Background

Mapping protein primary sequences to their three dimensional folds referred to as the 'second genetic code' remains an unsolved scientific problem. A crucial part of the problem concerns the geometrical specificity in side chain association leading to densely packed protein cores, a hallmark of correctly folded native structures. Thus, any model of packing within proteins should constitute an indispensable component of protein folding and design.

Results

In this study an attempt has been made to find, characterize and classify recurring patterns in the packing of side chain atoms within a protein which sustains its native fold. The interaction of side chain atoms within the protein core has been represented as a contact network based on the surface complementarity and overlap between associating side chain surfaces. Some network topologies definitely appear to be preferred and they have been termed 'packing motifs', analogous to super secondary structures in proteins. Study of the distribution of these motifs reveals the ubiquitous presence of typical smaller graphs, which appear to get linked or coalesce to give larger graphs, reminiscent of the nucleation-condensation model in protein folding. One such frequently occurring motif, also envisaged as the unit of clustering, the three residue clique was invariably found in regions of dense packing. Finally, topological measures based on surface contact networks appeared to be effective in discriminating sequences native to a specific fold amongst a set of decoys.

Conclusions

Out of innumerable topological possibilities, only a finite number of specific packing motifs are actually realized in proteins. This small number of motifs could serve as a basis set in the construction of larger networks. Of these, the triplet clique exhibits distinct preference both in terms of composition and geometry.     

Prediction of protein structure from ideal forms

For many years it has been accepted that the sequence of a protein can specify its three-dimensional structure. However, there has been limited progress in explaining how the sequence dictates its fold and no attempt to do this computationally without the use of specific structural data has ever succeeded for any protein larger than 100 residues. We describe a method that can predict complex folds up to almost 200 residues using only basic principles that do not include any elements of sequence homology. The method does not simulate the folding chain but generates many thousands of models based on an idealized representation of structure. Each rough model is scored and the best are refined. On a set of five proteins, the correct fold score well and when tested on a set of larger proteins, the correct fold was ranked highest for some proteins more than 150 residues, with others being close topological variants. All other methods that approach this level of success rely on the use of templates or fragments of known structures. Our method is unique in using a database of ideal models based on general packing rules that, in spirit, is closer to an ab initio approach.     

Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

When a protein exhibits complex kinetics of refolding, we often ascribe the complexity to slow isomerization events in the denatured protein, such as cis/trans isomerization of peptidyl prolyl bonds. Does the complex folding kinetics arise only from this well-known reason? Here, we have investigated the refolding of a proline-free variant of staphylococcal nuclease by stopped-flow, double-jump techniques, to examine the folding reactions without the slow prolyl isomerizations. As a result, the protein folds into the native state along at least two accessible parallel pathways, starting from a macroscopically single denatured-state ensemble. The presence of intermediates on the individual folding pathways has revealed the existence of multiple parallel pathways, and is characterized by multi-exponential folding kinetics with a lag phase. Therefore, a "single" amino acid sequence can fold along the multiple parallel pathways. This observation in staphylococcal nuclease suggests that the multiple folding may be more general than we have expected, because the multiple parallel-pathway folding cannot be excluded from proteins that show simpler kinetics.     

未折叠蛋白质的普遍初始热力学亚稳态

探索和理解蛋白质折叠问题一直是分子生物学、结构生物学和生物物理学的终极挑战.未折叠的蛋白质应该存在一种普遍初始热力学亚稳态,否则无法解释蛋白质是如何在剧烈的热振动干扰下完成快速精确折叠的.本文通过分析水溶液环境和蛋白质折叠的相关性,揭示了一种由水分子屏蔽效应引起的未折叠蛋白质的普遍初始热力学亚稳态,该亚稳态的存在是水溶液环境中水分子的物理性质决定,并赋予未折叠蛋白质抵抗热扰动和避免错误折叠的能力.我们通过研究已发表的实验数据和建立分子模型,找到了该初始热力学亚稳态存在的相关证据,并推测了该亚稳态导致蛋白质精确折叠的相关物理学机制.     

Determination of the folding of globular protein chain by the self-consistent field method

It is shown that and how it is possible to single out the chain fold which is thermo-dynamically most stable. The suggested approach is based on two physical ideas: A "molecular field" approximation permits to examine all protein structures which belong to the same "folding pattern". Only a limited set of the "potentially stable" folding patterns have to be examined. The general approach is illustrated by calculations of the stable folds for two beta-domains.     

Folding studies on a knotted protein

YibK is a 160 residue homodimeric protein belonging to the SPOUT class of methyltransferases. Proteins in this group all display a unique topological feature; the backbone polypeptide chain folds to form a deep trefoil knot. Such knotted structures were completely unpredicted, it being thought impossible for a protein to fold efficiently in this way. However, they are becoming more common and there are now a growing number of examples in the Protein Data Bank. These intriguing knotted structures represent a new and significant challenge in the field of protein folding. Here, we present an initial characterisation of the folding of YibK, one of the smallest knotted proteins to be identified. This is the first detailed folding study on a knotted protein to be reported. We have established conditions under which the protein can be denatured reversibly in vitro using urea, thereby showing that molecular chaperones are not required for the efficient folding of this protein. A series of equilibrium unfolding experiments were performed over a 400-fold range of protein concentration. Both secondary and tertiary structural probes show a single, protein concentration-dependent unfolding transition, and data are most consistent with a three-state equilibrium denaturation model involving a monomeric intermediate. Thermodynamic parameters obtained from the fit of the data to this model indicate that the intermediate is a stable species with appreciable secondary and tertiary structure; whether the topological knot remains in the intermediate state is still to be shown. Together, these results demonstrate that, despite its complex knotted structure, YibK is able to fold efficiently and behaves remarkably similarly to other dimeric proteins under equilibrium conditions.     

Strategies for Selection from Protein Libraries Composed of de Novo Designed Secondary Structure Modules

As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries (all alpha-helix, all beta-strand and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand and beta-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure alpha-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.     

Directed evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code

To better understand how amino acid sequences specify unique tertiary folds, we have used random mutagenesis and phage display selection to evolve proteins with a high degree of sequence identity but different tertiary structures (homologous heteromorphs). The starting proteins in this evolutionary process were the IgG binding domains of streptococcal protein G (G(B)) and staphylococcal protein A (A(B)). These nonhomologous domains are similar in size and function but have different folds. G(B) has an alpha/beta fold, and A(B) is a three-helix bundle (3-alpha). IgG binding function is used to select for mutant proteins which retain the correct tertiary structure as the level of sequence identity is increased. A detailed thermodynamic analysis of the folding reactions and binding reactions for a pair of homologous heteromorphs (59% identical) is presented. High-resolution NMR structures of the pair are presented by He et al. [(2005) Biochemistry 44, 14055-14061]. Because the homologous but heteromorphic proteins are identical at most positions in their sequence, their essential folding signals must reside in the positions of nonidentity. Further, the thermodynamic linkage between folding and binding is used to assess the propensity of one sequence to adopt two unique folds.     

Inter-residue interactions in protein folding and stability

During the process of protein folding, the amino acid residues along the polypeptide chain interact with each other in a cooperative manner to form the stable native structure. The knowledge about inter-residue interactions in protein structures is very helpful to understand the mechanism of protein folding and stability. In this review, we introduce the classification of inter-residue interactions into short, medium and long range based on a simple geometric approach. The features of these interactions in different structural classes of globular and membrane proteins, and in various folds have been delineated. The development of contact potentials and the application of inter-residue contacts for predicting the structural class and secondary structures of globular proteins, solvent accessibility, fold recognition and ab initio tertiary structure prediction have been evaluated. Further, the relationship between inter-residue contacts and protein-folding rates has been highlighted. Moreover, the importance of inter-residue interactions in protein-folding kinetics and for understanding the stability of proteins has been discussed. In essence, the information gained from the studies on inter-residue interactions provides valuable insights for understanding protein folding and de novo protein design.     

Why are both ends of the polypeptide chain on the outside of proteins?

Protein folding starts before the whole polypeptide has been synthesized by the ribosome. No matter how long the polypeptide is or how intricate the fold, both ends of the chain always end up on the surface. From a topological point of view, this is surprising; one would have expected to find the starting (N-terminal) end inside the core of the folded protein, just as in a ball of yarn. We suggest here that the reason for this apparent paradox is that the first amino acid of the emerging polypeptide chain is gripped during protein synthesis, perhaps by the ribosome, and is not released until the whole polypeptide has been synthesized. This binding would greatly decrease the degrees of freedom for the protein-folding process and could also explain why knots are so rare in proteins. Gripping would also guarantee that the N-terminal is accessible on the protein surface as required for binding of ubiquitin, which regulates the natural degradation of proteins and avoids buildup of protein aggregates, such as those found in Huntington's, Alzheimer's, Parkinson's, and other neurodegenerative diseases.     

A framework for describing topological frustration in models of protein folding

In a natively folded protein of moderate or larger size, the protein backbone may weave through itself in complex ways, raising questions about what sequence of events might have to occur in order for the protein to reach its native configuration from the unfolded state. A mathematical framework is presented here for describing the notion of a topological folding barrier, which occurs when a protein chain must pass through a hole or opening, formed by other regions of the protein structure. Different folding pathways encounter different numbers of such barriers and therefore different degrees of frustration. A dynamic programming algorithm finds the optimal theoretical folding path and minimal degree of frustration for a protein based on its natively folded configuration. Calculations over a database of protein structures provide insights into questions such as whether the path of minimal frustration might tend to favor folding from one or from many sites of folding nucleation, or whether proteins favor folding around the N terminus, thereby providing support for the hypothesis that proteins fold co-translationally. The computational methods are applied to a multi-disulfide bonded protein, with computational findings that are consistent with the experimentally observed folding pathway. Attention is drawn to certain complex protein folds for which the computational method suggests there may be a preferred site of nucleation or where folding is likely to proceed through a relatively well-defined pathway or intermediate. The computational analyses lead to testable models for protein folding.     

Directionality in protein fold prediction

Background  

Ever since the ground-breaking work of Anfinsen et al. in which a denatured protein was found to refold to its native state, it has been frequently stated by the protein fold prediction community that all the information required for protein folding lies in the amino acid sequence. Recent in vitro experiments and in silico computational studies, however, have shown that cotranslation may affect the folding pathway of some proteins, especially those of ancient folds. In this paper aspects of cotranslational folding have been incorporated into a protein structure prediction algorithm by adapting the Rosetta program to fold proteins as the nascent chain elongates. This makes it possible to conduct a pairwise comparison of folding accuracy, by comparing folds created sequentially from each end of the protein.     

Analysis of accessible surface of residues in proteins

We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent-accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein-accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and beta-structures (outside beta-sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from beta-sheets are the most inaccessible to solvent (10% accessible). Hence, beta-sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.     

Computational Modeling of Proteins based on Cellular Automata: A Method of HP Folding Approximation

The design of a protein folding approximation algorithm is not straightforward even when a simplified model is used. The folding problem is a combinatorial problem, where approximation and heuristic algorithms are usually used to find near optimal folds of proteins primary structures. Approximation algorithms provide guarantees on the distance to the optimal solution. The folding approximation approach proposed here depends on two-dimensional cellular automata to fold proteins presented in a well-studied simplified model called the hydrophobic–hydrophilic model. Cellular automata are discrete computational models that rely on local rules to produce some overall global behavior. One-third and one-fourth approximation algorithms choose a subset of the hydrophobic amino acids to form H–H contacts. Those algorithms start with finding a point to fold the protein sequence into two sides where one side ignores H’s at even positions and the other side ignores H’s at odd positions. In addition, blocks or groups of amino acids fold the same way according to a predefined normal form. We intend to improve approximation algorithms by considering all hydrophobic amino acids and folding based on the local neighborhood instead of using normal forms. The CA does not assume a fixed folding point. The proposed approach guarantees one half approximation minus the H–H endpoints. This lower bound guaranteed applies to short sequences only. This is proved as the core and the folds of the protein will have two identical sides for all short sequences.     

Look-up tables for protein solvent accessibility prediction and nearest neighbor effect analysis

We developed dictionaries of two-, three-, and five-residue patterns in proteins and computed the average solvent accessibility of the central residues in their native proteins. These dictionaries serve as a look-up table for making subsequent predictions of solvent accessibility of amino acid residues. We find that predictions made in this way are very close to those made using more sophisticated methods of solvent accessibility prediction. We also analyzed the effect of immediate neighbors on the solvent accessibility of residues. This helps us in understanding how the same residue type may have different accessible surface areas in different proteins and in different positions of the same protein. We observe that certain residues have a tendency to increase or decrease the solvent accessibility of their neighboring residues in C- or N-terminal positions. Interestingly, the C-terminal and N-terminal neighbor residues are found to have asymmetric roles in modifying solvent accessibility of residues. As expected, similar neighbors enhance the hydrophobic or hydrophilic character of residues. Detailed look-up tables are provided on the web at www.netasa.org/look-up/.     

Polymer Uncrossing and Knotting in Protein Folding,and Their Role in Minimal Folding Pathways

We introduce a method for calculating the extent to which chain non-crossing is important in the most efficient, optimal trajectories or pathways for a protein to fold. This involves recording all unphysical crossing events of a ghost chain, and calculating the minimal uncrossing cost that would have been required to avoid such events. A depth-first tree search algorithm is applied to find minimal transformations to fold , , , and knotted proteins. In all cases, the extra uncrossing/non-crossing distance is a small fraction of the total distance travelled by a ghost chain. Different structural classes may be distinguished by the amount of extra uncrossing distance, and the effectiveness of such discrimination is compared with other order parameters. It was seen that non-crossing distance over chain length provided the best discrimination between structural and kinetic classes. The scaling of non-crossing distance with chain length implies an inevitable crossover to entanglement-dominated folding mechanisms for sufficiently long chains. We further quantify the minimal folding pathways by collecting the sequence of uncrossing moves, which generally involve leg, loop, and elbow-like uncrossing moves, and rendering the collection of these moves over the unfolded ensemble as a multiple-transformation “alignment”. The consensus minimal pathway is constructed and shown schematically for representative cases of an , , and knotted protein. An overlap parameter is defined between pathways; we find that proteins have minimal overlap indicating diverse folding pathways, knotted proteins are highly constrained to follow a dominant pathway, and proteins are somewhere in between. Thus we have shown how topological chain constraints can induce dominant pathway mechanisms in protein folding.     

Influence of medium- and long-range interactions in different folding types of globular proteins

Recognition of protein fold from amino acid sequence is a challenging task. The structure and stability of proteins from different fold are mainly dictated by inter-residue interactions. In our earlier work, we have successfully used the medium- and long-range contacts for predicting the protein folding rates, discriminating globular and membrane proteins and for distinguishing protein structural classes. In this work, we analyze the role of inter-residue interactions in commonly occurring folds of globular proteins in order to understand their folding mechanisms. In the medium-range contacts, the globin fold and four-helical bundle proteins have more contacts than that of DNA-RNA fold although they all belong to all-alpha class. In long-range contacts, only the ribonuclease fold prefers 4-10 range and the other folding types prefer the range 21-30 in alpha/beta class proteins. Further, the preferred residues and residue pairs influenced by these different folds are discussed. The information about the preference of medium- and long-range contacts exhibited by the 20 amino acid residues can be effectively used to predict the folding type of each protein.     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).