Amino acid interaction preferences in helical membrane proteins

Membrane proteins are involved in a number of important biological functions. Yet, they are poorly understood from the structure and folding point of view. The external environment being drastically different from that of globular proteins, the intra-protein interactions in membrane proteins are also expected to be different. Hence, statistical potentials representing the features of inter-residue interactions based exclusively on the structures of membrane proteins are much needed. Currently, a reasonable number of structures are available, making it possible to undertake such an analysis on membrane proteins. In this study we have examined the inter-residue interaction propensities of amino acids in the membrane spanning regions of the alpha-helical membrane (HM) proteins. Recently we have shown that valuable information can be obtained on globular proteins by the evaluation of the pair-wise interactions of amino acids by classifying them into different structural environments, based on factors such as the secondary structure or the number of contacts that a residue can make. Here we have explored the possible ways of classifying the intra-protein environment of HM proteins and have developed scoring functions based on different classification schemes. On evaluation of different schemes, we find that the scheme which classifies amino acids to different intra-contact environment is the most promising one. Based on this classification scheme, we also redefine the hydrophobicity scale of amino acids in HM proteins.     

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.     

A search method for homologs of small proteins. Ubiquitin-like proteins in prokaryotic cells?

The question of protein homology versus analogy arises whenproteins share a common function or a common structural foldwithout any statistically significant amino acid sequence similarity.Even though two or more proteins do not have similar sequencesbut share a common fold and the same or closely related function,they are assumed to be homologs, descendant from a common ancestor.The problem of homolog identification is compounded in the caseof proteins of 100 or less amino acids. This is due to a limitednumber of basic single domain folds and to a likelihood of identifyingby chance sequence similarity. The latter arises from two conditions:first, any search of the currently very large protein databaseis likely to identify short regions of chance match; secondly,a direct sequence comparison among a small set of short proteinssharing a similar fold can detect many similar patterns of hydrophobicityeven if proteins do not descend from a common ancestor. In aneffort to identify distant homologs of the many ubiquitin proteins,we have developed a combined structure and sequence similarityapproach that attempts to overcome the above limitations ofhomolog identification. This approach results in the identificationof 90 probable ubiquitin-related proteins, including examplesfrom the two prokaryotic domains of life, Archaea and Bacteria. Received December 1, 2002; revised October 22, 2003; accepted October 24, 2003     

Heterogeneous-Backbone Foldamer Mimics of a Computationally Designed,Disulfide-Rich Miniprotein

Disulfide-rich peptides have found widespread use in the development of bioactive agents; however, low proteolytic stability and the difficulty of exerting synthetic control over chain topology present barriers to their application in some systems. Herein, we report a method that enables the creation of artificial backbone (“foldamer”) mimics of compact, disulfide-rich tertiary folds. Systematic replacement of a subset of natural α-residues with various artificial building blocks in the context of a computationally designed prototype sequence leads to “heterogeneous-backbone” variants that undergo clean oxidative folding, adopt tertiary structures indistinguishable from that of the prototype, and enjoy proteolytic protection beyond that inherent to the topologically constrained scaffold. Collectively, these results demonstrate systematic backbone substitution to be a viable method to engineer the properties of disulfide-rich sequences and expands the repertoire of protein mimicry by foldamers to an exciting new structural class.     

Recognition of analogous and homologous protein folds--assessment of prediction success and associated alignment accuracy using empirical substitution matrices

Fold recognition methods aim to use the information in the known protein structures (the targets) to identify that the sequence of a protein of unknown structure (the probe) will adopt a known fold. This paper highlights that the structural similarities sought by these methods can be divided into two types: remote homologues and analogues. Homologues are the result of divergent evolution and often share a common function. We define remote homologues as those that are not easily detectable by sequence comparison methods alone. Analogues do not have a common ancestor and generally do not have a common function. Several sets of empirical matrices for residue substitution, secondary structure conservation and residue accessibility conservation have previously been derived from aligned pairs of remote homologues and analogues (Russell et al., J. Mol. Biol., 1997, 269, 423-439). Here a method for fold recognition, FOLDFIT, is introduced that uses these matrices to match the sequences, secondary structures and residue accessibilities of the probe and target. The approach is evaluated on distinct datasets of analogous and remotely homologous folds. The accuracy of FOLDFIT with the different matrices on the two datasets is contrasted to results from another fold recognition method (THREADER) and to searches using mutation matrices in the absence of any structural information. FOLDFIT identifies at top rank 12 out of 18 remotely homologous folds and five out of nine analogous folds. The average alignment accuracies for residue and secondary structure equivalencing are much higher for homologous folds (residue approximately 42%, secondary structure approximately 78%) than for analogues folds (approximately 12%, approximately 47%). Sequence searches alone can be successful for several homologues in the testing sets but nearly always fail for the analogues. These results suggest that the recognition of analogous and remotely homologous folds should be assessed separately. This study has implications for the development and comparative evaluation of fold recognition algorithms.      

Sequence-structure specificity--how does an inverse folding approach work?

The inverse folding approach is a powerful tool in protein structure prediction when the native state of a sequence adopts one of the known protein folds. This is because some proteins show strong sequence- structure specificity in inverse folding experiments that allow gaps and insertions in the sequence-structure alignment. In those cases when structures similar to their native folds are included in the structure database, the z-scores (which measure the sequence-structure specificity) of these folds are well separated from those of other alternative structures. In this paper, we seek to understand the origin of this sequence-structure specificity and to identify how the specificity arises on passing from a short peptide chain to the entire protein sequence. To accomplish this objective, a simplified version of inverse folding, gapless inverse folding, is performed using sequence fragments of different sizes from 53 proteins. The results indicate that usually a significant portion of the entire protein sequence is necessary to show sequence-structure specificity, but there are regions in the sequence that begin to show this specificity at relatively short fragment size (15-20 residues). An island picture, in which the regions in the sequence that recognize their own native structure grow from some seed fragments, is observed as the fragment size increases. Usually, more similar structures to the native states are found in the top-scoring structural fragments in these high-specificity regions.      

The role of cotranslation in protein folding: a lattice model study

Computational studies of protein folding have implicitly assumed that folding occurs from a denatured state comprised of the entire protein. Cotranslational folding accounts for the linear production and release of a protein from the ribosome, allowing part of the protein to explore its conformation space before other parts have been synthesized. This gradual ‘extrusion’ from the ribosome can yield different folding kinetics than direct folding from the denatured state, for a lattice folding model. First, in model proteins containing chiefly short-ranged (local in sequence) contacts, cotranslational folding is shown to be significantly faster than direct folding from the denatured state. Secondly, for model proteins with two competing native states, cotranslational folding tilts the apparent equilibrium toward the state with a more local-contact dominant topology.     

3- Instead of 4-helix formation in a de novo designed protein in solution revealed by small-angle X-ray scattering

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so-called carboproteins. Here, we describe the preparation and structural studies of three alpha-helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4-alpha-helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small-angle X-ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1-helix folding topology in solution, while the free peptide formed a 3-helix bundle. This finding is consistent with the calculated alpha-helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4-helix carboprotein monomers with the 2x2-helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4-helix (3+1) carboproteins based on D-altropyranoside and D-galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance-geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability.     

Turncoat Polypeptides: We Adapt to Our Environment

A common interpretation of Anfinsen's hypothesis states that one amino acid sequence should fold into a single, native, ordered state, or a highly similar set thereof, coinciding with the global minimum in the folding-energy landscape, which, in turn, is responsible for the function of the protein. However, this classical view is challenged by many proteins and peptide sequences, which can adopt exchangeable, significantly dissimilar conformations that even fulfill different biological roles. The similarities and differences of concepts related to these proteins, mainly chameleon sequences, metamorphic proteins, and switch peptides, which are all denoted herein “turncoat” polypeptides, are reviewed. As well as adding a twist to the conventional view of protein folding, the lack of structural definition adds clear versatility to the activity of proteins and can be used as a tool for protein design and further application in biotechnology and biomedicine.     

The construction of new proteins

Recent progresses in the elucidation of the folding mechanism and topology of proteins revealed that the formation of folding units with specific topological features is not restricted to a unique primary sequence. This finding presents the basis for the design of polypeptides having the propensity to fold into a tertiary structure that can be achieved by the assembly of peptide blocks exhibiting stable secondary structures. Conformational studies on model peptides show that Aib containing peptides with chain-lengths of 12-15 residues are able to form stable amphiphilic helices in solution. On the other hand, oligopeptides with alternating hydrophilic and hydrophobic residues are capable for β-structure formation for chain-lengths of 6-8 residues. Those amphiphilic secondary structures have been used as building-blocks for the design and synthesis of artificial folding units, their amphiphilic nature acting as major driving force for intramolecular folding. Spectroscopic data obtained for two polypeptides designed as βαβ-models actually suggest a folded conformation of these molecules in aqueous solution. The implications of these findings for the design of biologically active folded polypeptides are discussed.     

Relationships between sequence and structure for the four-{alpha}-helix bundle tertiary motif in proteins

The sequences of four--helical bundle proteins are characterizedby a pattern of hydrophilic and hydrophobic amino acids whichis repeated every seven residues. At each position of the heptadrepeat there are specific constraints on the amino acid propertieswhich result from the topology of the tertiary motif. Theseconstraints give rise to patterns of amino acid distributionwhich are distinct from those of other proteins. The distributionsin each of the heptad positions have been determined by a statisticalanalysis of structural and sequence data derived from sevenfamines of aligned protein sequences. The constitution of eachposition is dominated by a very small number of different aminoacids, with the core positions consisting overwhelmingly ofLeu and Ala. The positional preferences of the individual aminoacids can be generally interpreted in terms of residue propertiesand topological constraints. The potential for four-a-helixbundle folding is reflected primarily in the pattern of residueoccurrence in the heptad and not in the overall amino acid compositionof the protein. Possible applications of this analysis in structurepredictions, sequence alignments and in the rational designand engineering of four-a-helkal bundle proteins are discussed.     

Structure-guided SCHEMA recombination of distantly related beta-lactamases

We constructed a library of beta-lactamases by recombining three naturally occurring homologs (TEM-1, PSE-4, SED-1) that share 34-42% sequence identity. Most chimeras created by recombining such distantly related proteins are unfolded due to unfavorable side-chain interactions that destabilize the folded structure. To enhance the fraction of properly folded chimeras, we designed the library using SCHEMA, a structure-guided approach to choosing the least disruptive crossover locations. Recombination at seven selected crossover positions generated 6561 chimeric sequences that differ from their closest parent at an average of 66 positions. Of 553 unique characterized chimeras, 111 (20%) retained beta-lactamase activity; the library contains hundreds more novel beta-lactamases. The functional chimeras share as little as 70% sequence identity with any known sequence and are characterized by low SCHEMA disruption (E) compared to the average nonfunctional chimera. Furthermore, many nonfunctional chimeras with low E are readily rescued by low error-rate random mutagenesis or by the introduction of a known stabilizing mutation (TEM-1 M182T). These results show that structure-guided recombination effectively generates a family of diverse, folded proteins even when the parents exhibit only 34% sequence identity. Furthermore, the fraction of sequences that encode folded and functional proteins can be enhanced by utilizing previously stabilized parental sequences.     

In search of the ideal protein sequence

The inverse of a folding problem is to find the ideal sequencethat folds into a particular protein structure. This problemhas been addressed using the topology fingerprintbased threadingalgorithm, capable of calculating a score (energy) of an arbitrarysequence-structure pair. At first, the search is conducted byunconstrained minimization of the energy in sequence space.It is shown that using energy as the only design criterion leadsto spurious solutions with incorrect amino acid composition.The problem lies in the general features of the protein energysurface as a function of both structure and sequence. The proposedsolution is to design the sequence by maximizing the differencebetween its energy in the desired structure and in other knownprotein structures. Depending on the size of the database ofstructures ‘to avoid’, sequences bearing significantsimilarity to the native sequence of the target protein areobtained using this procedure.     

Designability, aggregation propensity and duplication of disease-associated proteins

Over 2000 proteins in the Ensembl human genome database have been linked with disease information from OMIM. In comparison with all human proteins, we find that disease-associated proteins tend to have less designable folds in terms of their SCOP family counts, suggesting that they are intrinsically less robust to mutation and environmental stress. Disease proteins also tend to have isoelectric points closer to neutrality and more alternating hydrophilic-hydrophobic amino acid stretches compared with the average human protein. These results suggest that protein aggregation is a significant phenomenon associated with diseases. Another finding in this work is that many disease proteins are highly sequence similar to other disease proteins, suggesting that gene duplication has contributed to the expansion of disease-prone protein families.     

Identification and Molecular Cloning of Putative Odorant-Binding Proteins from the American Palm Weevil, Rhynchophorus palmarum L.

We have identified and cloned the cDNAs encoding two odorant-binding proteins (OBPs) from the American palm weevil (APW) Rhynchophorus palmarum (Coleoptera, Curculionidae). Degenerate primers were designed from the N-terminal sequences and were used in polymerase chain reaction (PCR) in order to obtain full-length sequences in both males and females. In both sexes, two different cDNAs were obtained, encoding 123 and 115 amino acid-deduced sequences. Each sequence showed few amino acid differences between the sexes. The proteins were named RpalOBP2 and RpalOBP4 for male, RpalOBP2' and RpalOBP4' for female, with the types 2 and 4 presenting only 34% identities. These proteins shared high identity with previously described coleopteran OBPs. In native gels, RpalOBP2 clearly separated into two bands and RpalOBP4 into three bands, suggesting the presence of several conformational isomers. Thus, OBP diversity in this species may rely on both the presence of OBPs from different classes and the occurrence of isoforms for each OBP.     

Protein design with L- and D-alpha-amino acid structures as the alphabet

Summarizing the implications of homochiral structures in interpeptide interactions, not only in the topology but also possibly in the physics of protein folding, this Account provides an overview of the concept of shape-specific protein design using D- and L-(alpha)amino acid structures as the alphabet. The molecular shapes accessible in de novo protein design are stereochemically defined. Indeed, the defining consideration for shape specificity in proteins to be alpha-helix/beta-sheet composites is the L configuration of the alpha-amino acid structures. The stereospecificity in shapes implies that protein shapes may be diversifiable stereochemically, that is, designable de novo, using D and L structures as the alphabet. Indeed, augmented with D enantiomers, Nature's alphabet will expand greatly in the diversity of polypeptide stereoisomers, for example, from 1(30) to 2(30)--that is, from one to ca. one billion--for a modestly sized 30-residue polypeptide. Furthermore, with each isomer having conformers stereospecific to its structure, molecular folds of specific shapes may be approachable sequentially when D and L structures are used as the alphabet. Illustrating the promise, 14-20-residue bracelet-, boat-, canoe-, and cup-shaped molecular folds were designed stereochemically or implemented as specific sequence plans in the D- and L-alpha-amino acid alphabet. In practical terms, canonical poly-L peptide folds were modified to the desired shapes via stereochemical mutations invoking enantiomer symmetries in the Ramachandran phi,psi space as the logic. For example, in designing the boat-shaped fold, the canonical beta-hairpin was reengineered in its flat planar structure via multiple coordinated L-to-D mutations in its position specific cross-strand neighbor residues, upturning its ends enclosing six side chains in a molecular cleft. While affirming the generality of the approach, the 20-residue molecular canoe and the 14-residue molecular cup are also presented as examples of the scope of functional design. The canoe, possessing alkali cation-specific catgrips in its main chain, and the cup, featuring an organic cation-specific aromatic triad in its side chains, do indeed display desired specificities in their ligand binding. Stereochemistry is, therefore, the crucial specifier of protein shapes and valuable as the tool for shape-specific protein design. Proteins in general, whether poly-L or mixed-D,L, require sequence effects of amino acid side chain structures for their stability, if not also for specifying them conformationally. The principles underlying these phenomena remain a puzzle, but studies invoking a stereochemical mutation approach to the problem have suggested that the poly-L structure may be crucial to the principles of sequential encoding of protein structures in amino acid side chains as the alphabet.     

Folding rate prediction based on neural network model

One of the most important challenges in biology is to understand the relationship between the folded structure of a protein and its primary amino acid sequence. A related and challenging task is to understand the relationship between sequences and folding rates of proteins. Previous studies found that one of contact order (CO), long-range order (LRO), and total contact distance (TCD) has a significant correlation with folding rate of protein. Although the predicted results from TCD can provide better results, the deviation is also large for some proteins. In this paper, we adopt back-propagation neural network to study the relationship between folding rate and protein structure. In our model, the input nodes are CO, LRO, and TCD, and the output node is folding rate. The number of nodes in the hidden layer is seven. Our results show that the relative errors for the predicted results are even lower than other methods in the literature. We also observe a best excellent correlation between the folding rate and contact parameters (including CO, LRO, and TCD), and find that the folding rate depends on CO, LRO and TCD simultaneously. This means that CO, LRO and TCD are similarly important in folding rate of protein. Some comparisons are made with other methods.