Characteristic features of amino acid residues in coiled-coil protein structures

Detailed analyses of protein structures provide an opportunity to understand conformation and function in terms of amino acid sequence and composition. In this work, we have systematically analyzed the characteristic features of the amino acid residues found in alpha-helical coiled-coils and, in so doing, have developed indices for their properties, conformational parameters, surrounding hydrophobicity and flexibility. As expected, there is preference for hydrophobic (Ala, Leu), positive (Lys, Arg) and negatively (Glu) charged residues in coiled-coil domains. However, the surrounding hydrophobicity of residues in coiled-coil domains is significantly less than that for residues in other regions of coiled-coil proteins. The analysis of temperature factors in coiled-coil proteins shows that the residues in these domains are more stable than those in other regions. Further, we have delineated the medium- and long-range contacts in coiled-coil domains and compared the results with those obtained for other (non-coiled-coil) parts of the same proteins and non-coiled-coil helical segments of globular proteins. The residues in coiled-coil domains are largely influenced by medium-range contacts, whereas long-range interactions play a dominant role in other regions of these same proteins as well as in non-coiled-coil helices. We have also revealed the preference of amino acid residues to form cation-pi interactions and we found that Arg is more likely to form such interactions than Lys. The parameters developed in this work can be used to understand the folding and stability of coiled-coil proteins in general.     

Exploring the environmental preference of weak interactions in (alpha/beta)8 barrel proteins

The environmental preference for the occurrence of noncanonical hydrogen bonding and cation-pi interactions, in a data set containing 71 nonredundant (alpha/beta)(8) barrel proteins, with respect to amino acid type, secondary structure, solvent accessibility, and stabilizing residues has been performed. Our analysis reveals some important findings, which include (a) higher contribution of weak interactions mediated by main-chain atoms irrespective of the amino acids involved; (b) domination of the aromatic amino acids among interactions involving side-chain atoms; (c) involvement of strands as the principal secondary structural unit, accommodating cross strand ion pair interaction and clustering of aromatic amino acid residues; (d) significant contribution to weak interactions occur in the solvent exposed areas of the protein; (e) majority of the interactions involve long-range contacts; (f) the preference of Arg is higher than Lys to form cation-pi interaction; and (g) probability of theoretically predicted stabilizing amino acid residues involved in weak interaction is higher for polar amino acids such as Trp, Glu, and Gln. On the whole, the present study reveals that the weak interactions contribute to the global stability of (alpha/beta)(8) TIM-barrel proteins in an environment-specific manner, which can possibly be exploited for protein engineering applications.     

To be folded or to be unfolded?

The lack of ordered structure in “natively unfolded” proteins raises a general question: Are there intrinsic properties of amino acid residues that are responsible for the absence of fixed structure at physiological conditions? In this article, we demonstrate that the competence of a protein to be folded or to be unfolded may be determined by the property of amino acid residues to form a sufficient number of contacts in a globular state. The expected average number of contacts per residue calculated from the amino acid sequence alone (using the average number of contacts for 20 amino acid residues in globular proteins) can be used as one of the simple indicators of natively unfolded proteins. The prediction accuracy for the sets of 80 folded and 90 natively unfolded proteins reaches 89% if the expected average number of contacts is used as a parameter and 83% in the case of hydrophobicity. An optimal set of artificial parameters for 20 amino acid residues obtained by Monte Carlo algorithm to maximally separate the sets of 90 natively unfolded and 80 folded proteins demonstrates the upper limit for prediction accuracy, which is 95%.     

Distribution of amino acid residues and residue-residue contacts in molecular chaperones.

The amino acid distribution and residue-residue contacts in molecular chaperones are different when compared to normal globular proteins. The study of molecular chaperones reveals a different surrounding environment to exist for the residues Cys, Trp, and His which may play an important role in determining the chaperone structures. Unlike globular proteins, it has been observed that a one-to-one correspondence between the amino acid distribution in a sequence and the structures of molecular chaperones. The preference of amino acid residues surrounding all 20 types of residues in secondary structures and their accessible surface areas have been analysed.     

Prognosis of the degree of exposure of amino acid residues in globular proteins in relation to various problems of protein engineering

The algorithm was developed to predict the degree of exposure of amino acid residues in globular proteins. This algorithm combined with standard discriminant analysis methods was used for evaluation of the accessibility of Lys and Arg residues for trypsin-like proteases attack. The procedure can be useful for a computer-aided design of prolonged-action protein drug preparations.     

Amino acid composition and thermal stability of proteins

This study investigates the relationship between the thermal stability of a globular protein and its amino acid composition. The method deals with the relationship between the amino acid compositions and melting points in a set of proteins by computing single-residue and group correlations. Groups of residues are shown to stabilize or destabilize the molecule against temperature. The stabilizing group consists of polar-charged residues and nonpolar residues possessing high surrounding hydrophobicity. The polar-uncharged residues destabilize the molecule against temperature, serine being the most destabilizing residue. A very high cooperativity exists among the stabilizing nonpolar residues suggesting that their characteristic clustering inside the globule may enhance the thermostability of a protein. In small globular proteins which act as single cooperative units, the melting temperature remains mainly a function of amino acid composition, whereas in complex molecules it depends on other factors also.     

Effect of spatial coupling of amino acid residues on the possibility of mutational substitution in globular proteins

Amino acid residues can be divided into similar groups by frequencies of interreplacements in the evolutionary pathway and by trends to spatial contacts at the tertiary structures of globular proteins. Each residue was compared to the cluster of spatial surrounding--the totality of residues spacially drawn together. 5210 clusters in 32 unhomologous proteins with established tertiary structure and 6447 clusters formed only by variables amino acid residues were analysed. Spatial contacts among residues were studied depending on the secondary structure and the amount of residues in a cluster. It was assumed that functionally admissible mutations may be defined, first of all, by the degree of neighboring of amino acid residues in the spatial surrounding.     

Structural analysis of residues involving cation-pi interactions in different folding types of membrane proteins

Cation-pi interactions play an important role to the stability of protein structures. In our earlier work, we have analyzed the influence and energetic contribution of cation-pi interactions in three-dimensional structures of membrane proteins. In this work, we investigate the characteristic features of residues that are involved in cation-pi interactions. We have computed several parameters, such as surrounding hydrophobicity, number of long-range contacts, conservation score and normalized B-factor for all these residues and identified their location, whether in the membrane or at surface. We found that the cation-pi interactions are mainly formed by long-range interactions. The cationic residues involved in cation-pi interactions have higher surrounding hydrophobicity than their average values in the whole dataset and an opposite trend is observed for aromatic residues. In transmembrane helical proteins, except Phe, all other residues that are responsible for cation-pi interactions are highly conserved with other related protein sequences whereas in transmembrane strand proteins, an appreciable conservation is observed only for Arg. The analysis on the flexibility of residues reveals that the cation-pi interaction forming residues are more stable than other residues. The results obtained in the present study would be helpful to understand the role of cation-pi interactions in the structure and folding of membrane proteins.     

Influence of cation-pi interactions on RNA-binding proteins

The energy contribution due to cation-π interactions has been computed for 37 RNA binding proteins. The contribution of these cation-π interacting residues in sequential separation, secondary structure involvement, solvent accessibility, and stabilization centers has been evaluated. Sequential separation of the cation-π involving residues show that, long range contacts predominates in all the proteins studied. Lys and Arg prefers to be in helical structures. Of the cation-π interacting residues, Arg and Lys were in the exposed regions and the aromatic residues (Phe, Tyr and Trp) were in the buried and partially buried regions in the protein structures. Stabilization centers for these proteins showed that all the five residues found in cation-π interactions are important in locating one or more of such centers. On the whole, the results presented in this work will be very useful for further investigations on the specificity and selectivity of RNA binding proteins and also for their structural studies.     

A 3D model of the voltage-dependent anion channel (VDAC)

Eukaryotic porins are a group of membrane proteins whose best known role is to form an aqueous pore channel in the mitochondrial outer membrane. As opposed to the bacterial porins (a large family of protein whose 3D structure has been determined by X-ray diffraction), the structure of eukaryotic porins (also termed VDACs, voltage-dependent anion-selective channels) is still a matter of debate. We analysed the secondary structure of VDAC from the yeast Saccharomyces cerevisiae, the fungus Neurospora crassa and the mouse with different types of neural network-based predictors. The predictors were able to discriminate membrane β-strands, globular -helices and membrane -helices and localised, in all three VDAC sequences, 16 β-strands along the chain. For all three sequences the N-terminal region showed a high propensity to form a globular -helix. The 16 β-strand VDAC motif was thus aligned to a bacterial porin-derived template containing a similar 16 β-strand motif. The alignment of the VDAC sequence with the bacterial porin sequence was used to compute a set of 3D coordinates, which constitutes the first 3D prediction of a eukaryotic porin. All the predicted structures assume a β-barrel structure composed of 16 β-strands with the N-terminus outside the membrane. Loops are shorter in this side of the membrane than in the other, where two long loops are protruding. The shape of the pore varies between almost circular for Neurospora and mouse and slightly oval for yeast. Average values between 3 and 2.5 nm at the C-carbon backbone are found for the diameter of the channels. In this model VDAC shows large portions of the structure exposed on both sides of the membrane. The architecture we determine allows speculation about the mechanism of possible interactions between VDAC and other proteins on both sides of the mitochondrial outer membrane. The computed 3D model is consistent with most of the experimental results so far reported.     

A rapid microbore HPLC method for determination of primary structure of β-lactoglobulin genetic variants

A rapid method for determination of the primary structures for β-lactoglobulin (β-LG) genetic variants is described. This included rapid microbore HPLC, amino acid analyses, and wherever necessary, direct peptide sequencing. Two novel variants of β-LG have been identified, bovine β-LG W and ovine β-LG C. The proteins were oxidized, digested with trypsin and separated using RP-HPLC. All peptides were recovered in a single run. Peptides with amino acid exchanges were identified by retention time and subjected to amino acid and sequence analyses. Ovine β-LG C differs from the ovine β-LG A variant by a single amino acid exchange at position 148 where Arg is replaced by Gln. Bovine β-LG W differs from bovine β-LG B by having Leu at position 56 instead of Ile. The method described here is reliable and can be used for mapping of 20–1000 pmol of material.     

Atom-wise statistics and prediction of solvent accessibility in proteins

In this work, we explore a novel method to broaden the scope of sequence-based predictions of solvent accessibility or accessible surface area (ASA) to the atomic level. All 167 heavy atoms from the 20 types of amino acid residues in proteins have been studied. An analysis of ASA distribution of these atomic groups in different proteins has been performed and rotamer-style libraries have been developed. We observe that the ASA of some atomic groups (e.g., backbone C and N atoms) can be estimated from the sequence environment within a mean absolute error of 2-3 angstroms(2). However, some side chain atoms such as CG in Pro, NH1 in Arg and NE2 in Gln show a strong variability making it more difficult to estimate their ASA from sequence environment. In general, the prediction of ASA becomes more difficult for atomic positions at the side chain extremities of long amino acid residues (aromatic side chain terminals being the exception). Several atomic groups are frequently exposed to solvent. Some of them have a bimodal distribution, suggesting two stable conformations in terms of their solvent exposure. More detailed understanding and prediction of solvent accessibility, i.e., at an atomic level is expected to help in bioinformatics approaches to structure prediction, functional relevance of atomic solvent accessibilities and other interaction analyses.     

Beta-strand interfaces of non-dimeric protein oligomers are characterized by scattered charged residue patterns

Protein oligomers are formed either permanently, transiently or even by default. The protein chains are associated through intermolecular interactions constituting the protein interface. The protein interfaces of 40 soluble protein oligomers of stœchiometries above two are investigated using a quantitative and qualitative methodology, which analyzes the x-ray structures of the protein oligomers and considers their interfaces as interaction networks. The protein oligomers of the dataset share the same geometry of interface, made by the association of two individual β-strands (β-interfaces), but are otherwise unrelated. The results show that the β-interfaces are made of two interdigitated interaction networks. One of them involves interactions between main chain atoms (backbone network) while the other involves interactions between side chain and backbone atoms or between only side chain atoms (side chain network). Each one has its own characteristics which can be associated to a distinct role. The secondary structure of the β-interfaces is implemented through the backbone networks which are enriched with the hydrophobic amino acids favored in intramolecular β-sheets (MCWIV). The intermolecular specificity is provided by the side chain networks via positioning different types of charged residues at the extremities (arginine) and in the middle (glutamic acid and histidine) of the interface. Such charge distribution helps discriminating between sequences of intermolecular β-strands, of intramolecular β-strands and of β-strands forming β-amyloid fibers. This might open new venues for drug designs and predictive tool developments. Moreover, the β-strands of the cholera toxin B subunit interface, when produced individually as synthetic peptides, are capable of inhibiting the assembly of the toxin into pentamers. Thus, their sequences contain the features necessary for a β-interface formation. Such β-strands could be considered as ‘assemblons’, independent associating units, by homology to the foldons (independent folding unit). Such property would be extremely valuable in term of assembly inhibitory drug development.     

BCL::Fold - De Novo Prediction of Complex and Large Protein Topologies by Assembly of Secondary Structure Elements

Computational de novo protein structure prediction is limited to small proteins of simple topology. The present work explores an approach to extend beyond the current limitations through assembling protein topologies from idealized α-helices and β-strands. The algorithm performs a Monte Carlo Metropolis simulated annealing folding simulation. It optimizes a knowledge-based potential that analyzes radius of gyration, β-strand pairing, secondary structure element (SSE) packing, amino acid pair distance, amino acid environment, contact order, secondary structure prediction agreement and loop closure. Discontinuation of the protein chain favors sampling of non-local contacts and thereby creation of complex protein topologies. The folding simulation is accelerated through exclusion of flexible loop regions further reducing the size of the conformational search space. The algorithm is benchmarked on 66 proteins with lengths between 83 and 293 amino acids. For 61 out of these proteins, the best SSE-only models obtained have an RMSD100 below 8.0 Å and recover more than 20% of the native contacts. The algorithm assembles protein topologies with up to 215 residues and a relative contact order of 0.46. The method is tailored to be used in conjunction with low-resolution or sparse experimental data sets which often provide restraints for regions of defined secondary structure.     

Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi,an Extremely Halophilic Microbe from Antarctica

The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.     

Influence of long-range contacts and surrounding residues on the transition state structures of proteins

Understanding the parameters influencing the formation of transition state structures in proteins is an important problem in protein folding and kinetics. In this work, we have analyzed the structure-based parameters, surrounding hydrophobicity, secondary structure, solvent accessibility, number of medium- and long-range contacts, and surrounding residues for understanding the transition state structures of 15 proteins. The analysis of Φ-values shows that 29% of the studied 378 mutants have a Φ-value of more than 0.5. The combination of different structure-based parameters could discriminate the residues that have a Φ-value cutoff of more than 0.5 with a 5-fold cross-validation accuracy of 68%, which indicates that the surrounding residues and contacts play important roles in the formation of transition state structures. Systematic analysis on different proteins reveals that the proteins azurin, cold shock protein, and C-terminal domain of ribosomal protein L9 are influenced by the number of medium- and long-range proteins, whereas barnase, FK506 binding protein, and IM9 are influenced by surrounding residues. The discrimination accuracy lies in the ranges of 81–95% and 74–85% for these respective classes of protein. Furthermore, the combination of surrounding residues and contacts improved the accuracy up to 24% in other considered proteins. We suggest that the structure-based parameters along with noncovalent interactions and conservation of residues may aid in identifying the potential residues in the formation of transition state structures in proteins.     

Search for structural factors responsible for the stability of proteins from thermophilic organisms

Database including 392 homologous pairs of proteins from thermophilic and mesophilic organisms was created. Using this database we have found that proteins from termophilic organisms contain more atom-atom contacts per residue in comparison with mesophilic homologues. Contribution to increase of the number of contacts gives exterior amino acid residues, accessible for the solvent. Amino acid composition of interior, inaccessible for the solvent, and exterior amino acid residues of proteins from thermophilic and mesophilic organisms were analyzed. We have obtained that exterior residues of proteins from thermophilic organisms contain more such amino acid residues as Lys, Arg and Glu and smaller such amino acid residues as Ala, Asp, Asn. Gln, Ser, and Thr in comparison with proteins from mesophilic organisms. Amino acid compositions of interior residues of considered proteins are not different.     

Protein Structures and Neutral Theory of Evolution

Abstract

The neutral theory of evolution is extended to the origin of protein molecules. Arguments are presented which suggest that the amino acid sequences of many globular proteins mainly represent “memorized” random sequences while biological evolution reduces to the “editing” these random sequences. Physical requirements for a functional globular protein are formulated and it is shown that many of these requirements do not involve strategical selection of amino acid sequences during biological evolution but are inherent also for typical random sequences. In particular, it is shown that random sequences of polar and unpolar amino acid residues can form α-helices and β-strands with lengths and arrangement along the chain similar to those in real globular proteins. These α- and β-regions in random sequences can form three-dimensional folding patterns also similar to those in real proteins. The arguments are presented suggesting that even the tight packing of side groups inside protein core do not require very strong biological selection of amino acid sequences either. Thus many structural features of real proteins can exist also in random sequences and the biological selection is needed mainly for the creation of active sites of proteins and for their stability under physiological conditions.