Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.     

Protein Flexibility Facilitates Quaternary Structure Assembly and Evolution

The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components.     

3D complex: a structural classification of protein complexes

Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.     

Classification of protein quaternary structure by functional domain composition

Background  

The number and the arrangement of subunits that form a protein are referred to as quaternary structure. Quaternary structure is an important protein attribute that is closely related to its function. Proteins with quaternary structure are called oligomeric proteins. Oligomeric proteins are involved in various biological processes, such as metabolism, signal transduction, and chromosome replication. Thus, it is highly desirable to develop some computational methods to automatically classify the quaternary structure of proteins from their sequences.     

Prediction of GPCRs with pseudo amino acid composition: employing composite features and grey incidence degree based classification

G-protein coupled receptor (GPCR) is a protein family that is found only in the Eukaryotes. They are used for the interfacing of cell to the outside world and are involved in many physiological processes. Their role in drug development is evident. Hence, the prediction of GPCRs is very much demanding. Because of the unavailability of 3D structures of most of the GPCRs; the statistical and machine learning based prediction of GPCRs is much demanding. The GPCRs are classified into family, sub family and sub-sub family levels in the proposed approach. We have extracted features using the hybrid combination of Pseudo amino acid, Fast Fourier Transform and Split amino acid techniques. The overall feature vector is then reduced using Principle component analysis. Mostly, GPCRs are composed of two or more sub units. The arrangement and number of sub units forming a GPCR are referred to as quaternary structure. The functions of GPCRs are closely related to their quaternary structure. The classification in the present research is performed using grey incidence degree (GID) measure, which can efficiently analyze the numerical relation between various components of GPCRs. The GID measure based classification has shown remarkable improvement in predicting GPCRs.     

Comparative analyses of quaternary arrangements in homo‐oligomeric proteins in superfamilies: Functional implications

A comprehensive analysis of the quaternary features of distantly related homo‐oligomeric proteins is the focus of the current study. This study has been performed at the levels of quaternary state, symmetry, and quaternary structure. Quaternary state and quaternary structure refers to the number of subunits and spatial arrangements of subunits, respectively. Using a large dataset of available 3D structures of biologically relevant assemblies, we show that only 53% of the distantly related homo‐oligomeric proteins have the same quaternary state. Considering these homologous homo‐oligomers with the same quaternary state, conservation of quaternary structures is observed only in 38% of the pairs. In 36% of the pairs of distantly related homo‐oligomers with different quaternary states the larger assembly in a pair shows high structural similarity with the entire quaternary structure of the related protein with lower quaternary state and it is referred as “Russian doll effect.” The differences in quaternary state and structure have been suggested to contribute to the functional diversity. Detailed investigations show that even though the gross functions of many distantly related homo‐oligomers are the same, finer level differences in molecular functions are manifested by differences in quaternary states and structures. Comparison of structures of biological assemblies in distantly and closely related homo‐oligomeric proteins throughout the study differentiates the effects of sequence divergence on the quaternary structures and function. Knowledge inferred from this study can provide insights for improved protein structure classification and function prediction of homo‐oligomers. Proteins 2016; 84:1190–1202. © 2016 Wiley Periodicals, Inc.     

Determinants of quaternary association in legume lectins

It is well known that the sequence of amino acids in proteins code for its tertiary structure. It is also known that there exists a relationship between sequence and the quaternary structure of proteins. The question addressed here is whether the nature of quaternary association can be predicted from the sequence, similar to the three-dimensional structure prediction from the sequence. The class of proteins called legume lectins is an interesting model system to investigate this problem, because they have very high sequence and tertiary structure homology, with diverse forms of quaternary association. Hence, we have used legume lectins as a probe in this paper to (1) gain novel insights about the relationship between sequence and quaternary structure; (2) identify the sequence motifs that are characteristic of a given type of quaternary association; and (3) predict the quaternary association from the sequence motif.     

Surface recognition elements of membrane protein oligomerization

Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts.     

Proportion of solvent-exposed amino acids in a protein and rate of protein evolution

Translational selection, including gene expression, protein abundance, and codon usage bias, has been suggested as the single dominant determinant of protein evolutionary rate in yeast. Here, we show that protein structure is also an important determinant. Buried residues, which are responsible for maintaining protein structure or are located on a stable interaction surface between 2 subunits, are usually under stronger evolutionary constraints than solvent-exposed residues. Our partial correlation analysis shows that, when whole proteins are included, the variance of evolutionary rate explained by the proportion of solvent-exposed residues (P(exposed)) can reach two-thirds of that explained by translational selection, indicating that P(exposed) is the most important determinant of protein evolutionary rate next only to translational selection. Our result suggests that proteins with many residues under selective constraint (e.g., maintaining structure or intermolecular interaction) tend to evolve slowly, supporting the "fitness (functional) density" hypothesis.     

Disulfide trapping of protein complexes on the yeast surface

Protein complexes are common in nature and play important roles in biology, but studying the quaternary structure formation in vitro is challenging since it involves lengthy and expensive biochemical steps. There are frequent technical difficulties as well with the sensitivity and resolution of the assays. In this regard, a technique that can analyze protein–protein interactions in high throughput would be a useful experimental tool. Here, we introduce a combination of yeast display and disulfide trapping that we refer to as stabilization of transient and unstable complexes by engineered disulfide (STUCKED) that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using fluorescence labeling. The technique uses an engineered intersubunit disulfide to covalently crosslink the subunits of a complex, so that the disulfide‐trapped complex can be displayed on the yeast surface for detection and analysis. Transient protein complexes are difficult to display on the yeast surface, since they may dissociate before they can be detected due to a long induction period in yeast. To this end, we show that three different quaternary structures with the subunit dissociation constant K_d ～ 0.5–20 µM, the antibody variable domain (Fv), the IL‐8 dimer, and the p53–MDM2 complex, cannot be displayed on the yeast surface as a noncovalent complex. However, when we introduce an interchain disulfide between the subunits, all three systems are efficiently displayed on the yeast surface, showing that disulfide trapping can help display protein complexes that cannot be displayed otherwise. We also demonstrate that a disulfide forms only between the subunits that interact specifically, the displayed complexes exhibit functional characteristics that are expected of wt proteins, the mutations that decrease the affinity of subunit interaction also reduce the display efficiency, and most of the disulfide stabilized complexes are formed within the secretory pathway during export to the surface. Disulfide crosslinking is therefore a convenient way to study weak protein association in the context of yeast display. Biotechnol. Bioeng. 2010; 106: 27–41. © 2009 Wiley Periodicals, Inc.     

Ion pairs in non-redundant protein structures

Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent-protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together. This paper deals with the possible ion pair combinations and networks in 25% and 90% non-redundant protein chains. Different types of ion pairs present in various secondary structural elements are analysed. The ion pairs existing between different subunits of multisubunit protein structures are also computed and the results of various analyses are presented in detail. The protein structures used in the analysis are solved using X-ray crystallography, whose resolution is better than or equal to 1.5 A and R-factor better than or equal to 20%. This study can, therefore, be useful for analyses of many protein functions. It also provides insights into the better understanding of the architecture of protein structure.     

Dynamic dissociating homo-oligomers and the control of protein function

Homo-oligomeric protein assemblies are known to participate in dynamic association/disassociation equilibria under native conditions, thus creating an equilibrium of assembly states. Such quaternary structure equilibria may be influenced in a physiologically significant manner either by covalent modification or by the non-covalent binding of ligands. This review follows the evolution of ideas about homo-oligomeric equilibria through the 20th and into the 21st centuries and the relationship of these equilibria to allosteric regulation by the non-covalent binding of ligands. A dynamic quaternary structure equilibria is described where the dissociated state can have alternate conformations that cannot reassociate to the original multimer; the alternate conformations dictate assembly to functionally distinct alternate multimers of finite stoichiometry. The functional distinction between different assemblies provides a mechanism for allostery. The requirement for dissociation distinguishes this morpheein model of allosteric regulation from the classical MWC concerted and KNF sequential models. These models are described alongside earlier dissociating allosteric models. The identification of proteins that exist as an equilibrium of diverse native quaternary structure assemblies has the potential to define new targets for allosteric modulation with significant consequences for further understanding and/or controlling protein structure and function. Thus, a rationale for identifying proteins that may use the morpheein model of allostery is presented and a selection of proteins for which published data suggests this mechanism may be operative are listed.     

A benchmark testing ground for integrating homology modeling and protein docking

Protein docking procedures carry out the task of predicting the structure of a protein–protein complex starting from the known structures of the individual protein components. More often than not, however, the structure of one or both components is not known, but can be derived by homology modeling on the basis of known structures of related proteins deposited in the Protein Data Bank (PDB). Thus, the problem is to develop methods that optimally integrate homology modeling and docking with the goal of predicting the structure of a complex directly from the amino acid sequences of its component proteins. One possibility is to use the best available homology modeling and docking methods. However, the models built for the individual subunits often differ to a significant degree from the bound conformation in the complex, often much more so than the differences observed between free and bound structures of the same protein, and therefore additional conformational adjustments, both at the backbone and side chain levels need to be modeled to achieve an accurate docking prediction. In particular, even homology models of overall good accuracy frequently include localized errors that unfavorably impact docking results. The predicted reliability of the different regions in the model can also serve as a useful input for the docking calculations. Here we present a benchmark dataset that should help to explore and solve combined modeling and docking problems. This dataset comprises a subset of the experimentally solved ‘target’ complexes from the widely used Docking Benchmark from the Weng Lab (excluding antibody–antigen complexes). This subset is extended to include the structures from the PDB related to those of the individual components of each complex, and hence represent potential templates for investigating and benchmarking integrated homology modeling and docking approaches. Template sets can be dynamically customized by specifying ranges in sequence similarity and in PDB release dates, or using other filtering options, such as excluding sets of specific structures from the template list. Multiple sequence alignments, as well as structural alignments of the templates to their corresponding subunits in the target are also provided. The resource is accessible online or can be downloaded at http://cluspro.org/benchmark , and is updated on a weekly basis in synchrony with new PDB releases. Proteins 2016; 85:10–16. © 2016 Wiley Periodicals, Inc.     

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI-MS and on protein structure under ESI-MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n-dodecyl-beta-D-glucopyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI-MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI-MS. However, several of the surfactants caused significant shifts in the charge-state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI-MS, can be useful for protein structure studies, if care is used in the interpretation of the results.     

Predicting protein quaternary structure by pseudo amino acid composition

In the protein universe, many proteins are composed of two or more polypeptide chains, generally referred to as subunits, that associate through noncovalent interactions and, occasionally, disulfide bonds. With the number of protein sequences entering into data banks rapidly increasing, we are confronted with a challenge: how to develop an automated method to identify the quaternary attribute for a new polypeptide chain (i.e., whether it is formed just as a monomer, or as a dimer, trimer, or any other oligomer). This is important, because the functions of proteins are closely related to their quaternary attribute. For example, some critical ligands only bind to dimers but not to monomers; some marvelous allosteric transitions only occur in tetramers but not other oligomers; and some ion channels are formed by tetramers, whereas others are formed by pentamers. To explore this problem, we adopted the pseudo amino acid composition originally proposed for improving the prediction of protein subcellular location (Chou, Proteins, 2001; 43:246-255). The advantage of using the pseudo amino acid composition to represent a protein is that it has paved a way that can take into account a considerable amount of sequence-order effects to significantly improve prediction quality. Results obtained by resubstitution, jack-knife, and independent data set tests, have indicated that the current approach might be quite promising in dealing with such an extremely complicated and difficult problem.     

The 2.2 A resolution structure of RpiB/AlsB from Escherichia coli illustrates a new approach to the ribose-5-phosphate isomerase reaction

Ribose-5-phosphate isomerases (EC 5.3.1.6) interconvert ribose 5-phosphate and ribulose 5-phosphate. This reaction permits the synthesis of ribose from other sugars, as well as the recycling of sugars from nucleotide breakdown. Two unrelated types of enzyme can catalyze the reaction. The most common, RpiA, is present in almost all organisms (including Escherichia coli), and is highly conserved. The second type, RpiB, is present in some bacterial and eukaryotic species and is well conserved. In E.coli, RpiB is sometimes referred to as AlsB, because it can take part in the metabolism of the rare sugar, allose, as well as the much more common ribose sugars. We report here the structure of RpiB/AlsB from E.coli, solved by multi-wavelength anomalous diffraction (MAD) phasing, and refined to 2.2A resolution. RpiB is the first structure to be solved from pfam02502 (the RpiB/LacAB family). It exhibits a Rossmann-type alphabetaalpha-sandwich fold that is common to many nucleotide-binding proteins, as well as other proteins with different functions. This structure is quite distinct from that of the previously solved RpiA; although both are, to some extent, based on the Rossmann fold, their tertiary and quaternary structures are very different. The four molecules in the RpiB asymmetric unit represent a dimer of dimers. Active-site residues were identified at the interface between the subunits, such that each active site has contributions from both subunits. Kinetic studies indicate that RpiB is nearly as efficient as RpiA, despite its completely different catalytic machinery. The sequence and structural results further suggest that the two homologous components of LacAB (galactose-6-phosphate isomerase) will compose a bi-functional enzyme; the second activity is unknown.     

A comprehensive analysis of non-sequential alignments between all protein structures

Background  

The majority of relations between proteins can be represented as a conventional sequential alignment. Nevertheless, unusual non-sequential alignments with different connectivity of the aligned fragments in compared proteins have been reported by many researchers. It is interesting to understand those non-sequential alignments; are they unique, sporadic cases or they occur frequently; do they belong to a few specific folds or spread among many different folds, as a common feature of protein structure. We present here a comprehensive large-scale study of non-sequential alignments between available protein structures in Protein Data Bank.     

Bioinformatics analyses of circular dichroism protein reference databases

MOTIVATION: Circular dichroism (CD) spectroscopy has become established as a key method for determining the secondary structure contents of proteins which has had a significant impact on molecular biology. Many excellent mathematical protocols have been developed for this purpose and their quality is above question. However, reference database sets of proteins, with CD spectra matched to secondary structure components derived from X-ray structures, provide the key resource for this task. These databases were created many years ago, before most CD spectrophotometers became standardized and before it was commonplace to validate X-ray structures prior to publication. The analyses presented here were undertaken to investigate the overall quality of these reference databases in light of their extensive usage in determining protein secondary structure content from CD spectra. RESULTS: The analyses show that there are a number of significant problems associated with the CD reference database sets in current use. There are disparities between CD spectra for the same protein collected by different groups. These include differences in magnitudes, peak positions or both. However, many current reference sets are now amalgamations of spectra from these groups, introducing inconsistencies that can lead to inaccuracies in the determination of secondary structure components from the CD spectra. A number of the X-ray structures used fall short on the validation criteria now employed as standard for structure determination. Many have substantial percentages of residues in the disallowed regions of the Ramachandran plot. Hence their calculated secondary structure components, used as a foundation for the reference databases, are likely to be in error. Additionally, the coverage of secondary structure space in the reference datasets is poorly correlated to the secondary structure components found in the Protein Data Bank. A conclusion is that a new reference CD database with cross-correlated, machine-independent CD spectra and validated X-ray structures that cover more secondary structure components, including diverse protein folds, is now needed. However, that reasonably accurate values for the secondary structure content of proteins can be determined from spectra is a testament to CD spectroscopy being a very powerful technique.