Biomolecular Forms and Functions

The following sections are included:

• PREFACE

• CONTENTS

G. N. Ramachandran is among the founding fathers of structural molecular biology. He made pioneering contributions in computational biology, modelling and what we now call bioinformatics. The triple helical coiled coil structure of collagen proposed by him forms the basis of much of collagen research at the molecular level. The Ramachandran map remains the simplest descriptor and tool for validation of protein structures. He has left his imprint on almost all aspects of biomolecular conformation. His contributions in the area of theoretical crystallography have been outstanding. His legacy bas provided inspiration for the further development of structural biology in India. After a pause, computational biology and bioinformatics are in a resurgent phase. One of the two schools established by Ramachandran pioneered the development of macromolecular crystallography, which has now grown into an important component of modern biological research in India. Macromolecular NMR studies in the country are presently gathering momentum. Structural biology in India is now poised to again approach heights of the kind that Ramachandran conquered more than a generation ago.

Collagen, a protein found ubiquitously in vertebrate and invertebrate species, has a triple-helical structure that has been historically elucidated through fiber studies. More recently, single crystal X-ray experiments have corroborated, expanded upon, and clarified what was known from the earlier studies. Here we offer a survey of the triple-helical entries present in the Protein Data Bank (PDB) and a discussion of how these studies have provided new insight into the hydrogen bonding patterns, atomic structure, hydration and helical symmetry of the collagen molecule.

The Ramachandran Plot, first introduced half a century ago, maps the backbone torsion angles – ϕ and ψ – for a dipeptide, showing that steric restrictions eliminate approximately ¾ of conceivable conformational space. Titis chapter revisits and then generalizes this early result, a follow-on to ideas implicit in the Plot. Ramachandran's dramatic result notwithstanding, subsequent work seemed to indicate that dipeptide restrictions have a negligible impact on overall protein conformation. Consequently, later decades witnessed an ever-increasing emphasis on the significance of attractive forces in folding simulations, with concomitant neglect of the organizing power of excluding forces. Attractive forces act by stabilizing native interactions, while excluding forces such as steric clashes act by destabilizing non-native interactions. This chapter summarizes some recent advances that pertain to both types of excluding forces: (a) systematic local steric clashes beyond the dipeptide and (b) the strongly destabilizing influence of buried polar groups that lack hydrogen bond partners.

The scientific history of the Ramachandran plot is reviewed, emphasizing relationships to the title theme and to trends in current research. The growth and quality of macromolecular structure data have enabled the understanding of relationships with further variables and the application to an ever-widening set of uses such as prediction, simulation, design, motif identification, and structure validation and improvement. Then a current research example is explored, using a new dataset of 8000 selected protein chains and <1.5 million quality-filtered residues. The new Ramachandran plots show an unprecedented level of detail, allow the valid addition of more subcategories and more dimensions, and point the way toward the feasibility of an essentially complete and robust treatment of torsional conformation in the near future.

The Ramachandran plot has been the mainstay of protein structure validation for many years. Its detailed structure has been continually analysed and refined as more and more experimentally determined models of protein 3D structures have become available, particularly at high and ultra-high resolution. As more data has accumulated, so the differences in the plot for different amino acids, and with different residue neighbours, or in different structural contexts, have become apparent. Validation software has had to adapt to these subtle changes and a single Ramachandran plot can no longer accurately depict ‘favourable’ and ‘disallowed’ regions for all amino acid types.

The Ramachandran map consisting of the dihedral angles ϕ and ψ has been an object of study for 50 years, since its presentation by Ramachandran, Ramakrishnan, and Sasisekharan in July 1963. As the number of structures has grown, it has become possible to apply modem non-parametric statistics to develop proper probability density estimates for the Ramachandran distribution for each amino acid type and different input data sets. It has also become possible to derive classification functions and regressions as a function of ϕ and ψ. For example our most recent backbone-dependent rotamer library consists of the classification probability of the side-chain rotamers and a regression of the mean dihedral angles and variances. The backbone and side-chain bond angles of amino acids also vary with ϕ, ψ, as observed in non-parametric regressions from sub-Ångstrom crystal structure data. This variation confirms Ramachandran's early work that some regions of the Ramachandran map are only accessible with larger values of the backbone bond angle N-Cκ-C. Most non-parametric statistical methods can be appropriately modified for pairs of angles and applied to the Ramachandran variables, including kernel density estimates and kernel regressions with the bivariate von Mises distribution, hierarchical Dirichlet processes, and Gaussian processes.

The allowed and the “disallowed” regions in the celebrated Ramachandran map ([ϕ-ψ] map) was elegantly deduced by Ramachandran, Ramakrishnan and Sasisekharan even before the protein crystal structures became available. This powerful map was derived based on rigid geometry of the peptide group and later several investigations on protein crystal structures reported the occurrence of a small fraction of the [ϕ-ψ] torsion angles in the disallowed region. The question is what factors make these residues adopt disallowed conformation? Is it driven by the necessity to maintain the overall topology or is it associated with function or is it just that the disallowed conformations are extreme limits of the allowed conformations? Today, with the availability of a large number of high resolution crystal structures, we have revisited this problem. Apart from validating some of the earlier findings such as residue propensities, preferred location in the secondary structure, we have explored their spatial neighborhood preferences using the protein structure network [PSN] approach developed in our lab. Finally, the structural and functional implications of the disallowed conformati0118 are examined.

Similarity or variability of protein structures is usually measured by superposition of structures and then assessed by root mean square deviation of the superposed main chain atoms. The relationship between equivalent main chain atoms (or Cα atoms) and the variability in their dihedral angles has been assessed in this work. Proteins with identical sequences (or point mutants), those with homologous structures, and those whose structures have been determined by NMR were used to assess such relationship.

A regular secondary structure is described by a well defined set of values for the backbone dihedral angles (φ, ψ and ω) in a polypeptide chain. However in real protein structures small local variations give rise to distortions from the ideal structures, which can lead to considerable variation in higher order organization. Protein structure analysis and accurate assignment of various structural elements, especially their terminii, are important first step in protein structure prediction and design. Various algorithms are available for assigning secondary structure elements in proteins but some lacunae still exist. In this study, results of a recently developed in-house program ASSP have been compared with those from STRIDE, in identification of α-helical regions in both globular and membrane proteins. It is found that, while a combination of hydrogen bond pattern and backbone torsional angles (φ-ψ) are generally used to define secondary structure elements, the geometry of the C^α atom trace by itself is sufficient to define the parameters of helical structures in proteins. It is also possible to differentiate the various helical structures by their C^α trace and identify the deviations occurring both at mid-positions as well as at the terminii of α-helices, which often lead to occurrence of 3₁₀ and π-helical fragments in both globular and membrane proteins.

Using a dataset of 1164 crystal structures of largely non-homologous proteins defined at a resolution of 1.5Å or better, we have investigated the (ϕ, ψ) preferences of 20 residue types by considering the residues which occur in loops. Propensities of residue types to occur in the loops with (ϕ, ψ) values in the α_R region of the Ramachandran map has a poor correlation coefficient of 0.48 to the Chou-Fasman propensities of the residue types to occur in the κ-helical segments. However the correlation coefficient between propensities of residues in loops to adopt β conformations and those in β-sheet is much higher (0.95). These observations suggest that α-helix formation is well influenced by the local amino acid sequence while intrinsic preference of residue types for β-sheet plays a major role in the formation of β-sheet. The main chain polar groups of residues in loops, that can affect the (ϕ, ψ) values, can be involved in intra-molecular hydrogen bonding. Therefore we investigated further by considering subset of residues in loops with low (0 to 2) number of intra-molecular hydrogen bonds per residue involving main chain polar atoms. For this subset, the correlation coefficients between propensities for α-helix and α_R region and between β-sheet and β-region are 0.26 and 0.64 respectively. This reiterates higher intrinsic tendency of β-region favouring residues to adopt β-sheet than α_R region favouring residues to adopt α-helical structure.

The conformations available to polypeptides are determined by the interatomic forces acting on the peptide units, whereby backbone torsion angles are restricted as described by the Ramachandran plot. Although typical proteins are composed predominantly from α-helices and β-sheets, they nevertheless adopt diverse tertiary structure, each folded as dictated by its unique amino-acid sequence. Despite such uniqueness, however, the functioning of many proteins involves changes between quite different conformations. The study of large-scale conformational changes, particularly in large systems, is facilitated by a coarse-grained representation such as provided by virtually bonded Cα atoms. We have developed a virtual atom molecular mechanics (VAMM) force field to describe conformational dynamics in proteins and a VAMM-based algorithm for computing conformational transition pathways. Here we describe the stereochemical analysis of proteins in this coarse-grained representation, comparing the relevant plots in coarse-grained conformational space to the corresponding RamaChandran plots, having contoured each at levels determined statistically from residues in a large database. The distributions shown for an all-α protein, two all-β proteins and one α+β protein serve to relate the coarse-grained distributions to the familiar Ramachandran plot.

The central role of multiprotein assemblies in living organisms makes them attractive targets for therapeutic intervention. Here, we briefly describe the classification of protein complexes and the available databases for their study. The chapter explores the Current findings for peptide and small molecule binding to protein interfaces and presents insights for modulating the protein-protein interactions with synthetic agents.

A docking method, surFit, was developed that automatically and semi-automatically docks a pair of protein molecular surfaces. It performs the following six procedures: Binding site prediction, Rigid surface docking, Coarse scoring, Refinement, Precise scoring, and Re-refinement by molecular dynamics simulation. The first four procedures have been automated and implemented in the webserver surFit. The current protocol successfully built many acceptable predicted complex structures with high qualities for the recent CAPRI targets, and accurately estimated the solvent water positions at the interface for CAPRI Target 47. To reveal the complex structure of an intrinsically disordered protein (IDP) with its partner receptor protein, enhanced sampling computations were performed to simulate the free energy landscapes of the IDP with and without the receptor. Consequently, both induced fitting and population shift mechanisms were observed for the NRSF-Sin3 system.

Among the fundamental issues critical for body's defense is the maintenance of self-nonself discrimination. How does the immune system handle a limitless antigenic space and efficiently discriminate within a biological energy ‘budget’? Is there a unique antibody for every new antigen that is encountered? Are there strategies built into the immune system to cope with the pathogenic intelligence? Crystallographic studies on antibody diversity have given newer insights into the mechanism of repertoire amplification at the germ line stage offering interesting physiological perspectives on primary immune response. Structural data pertaining to the interactions between germline antibodies and their corresponding antigens have been analysed. These studies redefine antigen recognition within the established norms of structural chemistry bringing out intriguingly new aspects of antigen recognition in humoral antibody response.

Nuclear Magnetic Resonance (NMR) spectroscopy has been undergoing unparalleled development, much of which has been driven by applications in structural biology, especially, proteins. We have designed new NMR methods and protocols for rapid chemical shift assignment of protein backbone resonances. These have enabled rapid determination of protein structures, elucidation of folding transitions, and getting valuable insights into protein-protein interaction and self association. These have significant implications for understanding structure-dynamics-function relationships, mechanisms of protein mis-folding, and thus may help in designing of drugs against specific diseases.

Understanding the recognition mechanism of proteins with other molecules is a challenging task in molecular and computational biology. Experiments provide a wealth of data on binding specificity in terms of dissociation constant and binding free energy change upon protein complex formation. Computationally, the three-dimensional structures of protein complexes have been used to delineate the binding sites and to understand their recognition mechanisms. We have developed an unified energy based approach for identifying the binding site residues in protein–protein, protein-RNA and protein-DNA complexes. In protein-protein complexes, the residues and residue-pairs with charged and aromatic side chains are found to be important for binding. These residues influence to form cation-π, electrostatic and aromatic interactions. In protein RNA complexes, the positively charged, polar and aromatic residues are important for binding. These residues influence to form electrostatic, hydrogen bonding and stacking interactions. The positive charged and polar residues prefer to bind with DNA in protein-DNA complexes. Our observations showed a good agreement with the experimental binding specificity of protein-protein and protein-nucleic acid complexes.

We discuss the recent extraction of signatures of stoichiometry driven universal spatial organization of backbones of folded proteins regardless of their size, shape/structure and function. We present further evidence for secularity of amino acids in protein structures from the perspectives of surface area and energy. While conceptual fragmentation to gain insights into the diversity of protein structures appears to be a popular approach. we believe that the secrets to solving the protein folding problem lie in appreciating concepts that are universally applicable.

The cold-shock domain (CSD) is a structurally conserved nucleic acid-binding domain that is present in all kingdoms of life from bacteria to vertebrates. It associates with a wide range of single-stranded nucleic acids via a conserved, hydrophobic platform located on one side of its β-barrel fold. The limited sequence specificity in nucleic-acid binding, in line with its RNA-remodeling function, makes the CSD a powerful molecular module for regulating RNA metabolism at various levels. Moreover, combinations of CSDs with other protein domains result in an enormous functional versatility of CSD-containing proteins that comprises cold acclimation in bacteria as well as regulation of development, differentiation and stress tolerance in higher eukaryotes. This review summarizes the role of CSDs in prokaryotic and eukaryotic proteins with a special emphasis on their nucleic acid-binding and domain-swap capabilities.

Many proteins essential for the viability of the cell induce large-scale deformations in the DNA double helix. Some of these deformations include transitions of regular B DNA to alternate helical forms, such as A DNA, where base pairs are unwound, displaced, and inclined with respect to the helical axis. Here we examine structural features of the A-like DNA base-pair steps found in a diverse set of high-resolution protein-DNA complexes. The protein-bound DNA is more deformable than the A DNA crystallized in the absence of protein, and the protein microenvironment surrounding A DNA differs in subtle ways from that associated with other types of DNA The kinds of amino-acid atoms in immediate contact with the sugars, phosphates, and bases hint of ways in which small molecules may cluster around A DNA in solution and how changes in solvent may contribute to alterations of helical structure.

The biological macromolecular world is chiral. How homochirality at the molecular level crept into the living system is an aspect which still remains unresolved. The presence of D-amino acids in organisms apart from bacteria was not appreciated until recently. Several studies in the last three decades have established the ubiquity of D-amino acids in the living world. Some of these D-amino acids have now been implicated in several important physiological functions, including ncurotransmission and hormone regulation. However, proteins are comprised of only L-amino acids; a protein containing both L- and D-enantiomers loses its functionality owing to the fact that it fails to take its proper conformation. Hence, it becomes imperative for the cell to maintain “enantiomeric fidelity” during every step of translation. To this end, the cell employs several molecular machineries which, in their own capacity, help in excluding D-amino acids from polypeptides. An interesting case is presented by D-aminoacy1-tRNA deacylase which removes a D-amino acid that is mischarged onto the corresponding tRNA. Further discrimination against D-amino acids at the level of elongation factors and ribosomes has been noted. All these cellular systems play their part in perpetuation of homochirality in proteins.

Folding into compact globular structures, with well-defined modules of secondary structure, appears to be a characteristic of long polypeptide chains, with a specific patterning of coded amino acid residues along the length of sequence. Cooperative hydrogen bond driven secondary structure formation and solvent forces, which contribute favorably to the entropy of folding, by promoting compaction of the polymeric chain, have long been discussed as major determinants of the folding process. First principles design approaches, which use non-coded amino acids, employ an alternative structure directing strategy, by using amino acid residues which exhibit a strong conformational bias for specific regions of the Ramachandran map. This overview of ongoing studies in the authors' laboratory, attempts to explore the use of conformationally restricted amino acid residues in the design of peptides with well-defined secondary structures. Short peptides composed of 20 genetically coded amino acids usually exist in solution as an ensemble of equilibrating conformations. Apolar peptide sequences, which are readily soluble in organic solvents like chloroform and methanol, facilitate formation of structures which are predominately driven by intramolecular hydrogen bond formation. The choice of sequences containing residues with a limited range of conformational choices strongly favors formation of local turn structures, stabilized by short range intramolecular hydrogen bonds. Two residue β-turns can nucleate either helical or hairpin folding, depending on the precise conformation of the tum segment Restriction of the conformational space available to amino acid residues is easily achieved by introduction of an additional alkyl group at the Cα. carbon atom or by side chain backbone cyclization, as in proline. Studies of synthetic sequences incorporating two prototype residues α-aminoisobutyric acid (Aib) and D-proline (DPro) illustrate the utility of the strategy in construction of helices and hairpins. Extensions to the design of conformationally switchable sequences and structurally defined hybrid peptides containing backbone homologated residues are also surveyed.

β-amino acids are naturally occurring non-protein amino acids found in several fungal peptide antibiotics. β-amino acids can also tailor stable well-defined secondary structures in peptides. Cyclic β-amino acids can restrain peptide conformations further as the C² and C³ bond is now part of a ring. Data analysis of the crystal structures of these β-amino acids, both for cyclic as well as acyclic amino acid residues, from the 2012 Cambridge structure database reveals that the (φ, ψ) values are spread in the allowed region of the glycine Ramachandran map.

The significant contribution of naturally occurring disulfide bonds to protein stability bas encouraged development of methods to engineer non-native disulfides in proteins. These have yielded mixed results. We summarize applications of the program MODIP for disulfide engineering. The program predicts sites in proteins where disulfides can be stably introduced. The program has also been used as an aid in conformational analysis of naturally occurring disulfides in α-helices, antiparallel and parallel β-strands. Disulfides in α-helices occur only at N-termini, where the first cysteine residue is the N-cap residue of the helix. The disulfide occurs as a CXXC motif and can possess redox activity. In antiparallel β-strands, disulfides occur exclusively at non-hydrogen bonded (NHB) registered pairs of antiparallel β-sheets with only 1 known natural example occurring at a hydrogen bonded (HB) registered pair. Conformational analysis suggests that disulfides between HB residue pairs are under torsional strain. A similar analysis to characterize disulfides in parallel β-strands was carried out. We observed that only 9 instances of cross-strand disulfides exist in a non-redundant dataset. Stereochemical analysis shows that while the χ angles are similar to those of other disulfides, the χ¹ and χ¹ angles show more variation and that one of the strands is generally an edge strand.

A strongly twisted and coiled β-hairpin can be represented as a double-helical structure in which the strands are twisted and coiled in a right-handed sense. The double superhelix has both a concave and a convex surface. A distinctive feature of this structure is that it is always formed by the right-turned β-hairpin when viewed from the concave side of the superhelix. Another feature of this structure is that the cp, φ, ψ values alternate along the polypeptide chain and cp, φ, ψ values of the “inside” residues located on the concave surface have a strong tendency to fall in the β_E-region (extended conformations) and those of the “outside” residues in the β_p-region (polyproline region) of the Ramachandran map. A stereochemical analysis shows that glycines in “inside” and prolines in “outside” positions have to facilitate formation of coiled β-hairpins. Statistical data confirm these conclusions. Thus, the strongly twisted and coiled β-hairpin has the unique fold itself and can be taken as the starting structure in modeling of protein folds and folding pathways. The larger protein folds are obtained by stepwise addition of secondary structural elements to the root β-hairpin in accordance with a set of simple rules. A structural tree constructed using this approach includes 112 non-homologous proteins classified as a subclass of (α + β)-proteins and referred to as wrap-proteins.

The phenomenon of protein domain swapping, observed originally amongst a handful of protein examples, is acquiring attention not only due to statistically significant number of examples, but their relevance in neurodegenerative and other diseases. We describe an algorithm which could automatically detect ‘hinge regions’ in 87% of domain swapped examples, using structural properties such as higher inter-chain interactions, solvent accessibility and unstructured “coil” backbone conformation. This has enabled us to update our 3DSwap database which now contains data for 2057 protein structural entries where we observe swapping. Conformational analysis of four-residue hinge regions, in comparison with the equivalent region in tbe non-swapped domain counterpart, reveals fairly extended conformations are adopted by short hinges. Starting from one such hinge region, using metadynamics simulations, we show that there is a strong tendency to adopt to the more popular β-turn conformation as observed in tbe non-swapped domain structure. More detailed investigations of the inherently flexible, yet stable multiple conformations of the hinge regions that underlie the domain swapping structural transitions will be valuable.

Almost a decade ago, the widely accepted and acknowledged sequence-structure-function paradigm faced a major challenge. Proteins were believed to be functional only when in structured/folded state with a well-defined and stable three dimensional conformation. Denatured proteins were found to loose function upon losing their structure. Though there were some indications about flexibility linked functionality of the proteins, it largely remained unnoticed till the beginning of this millennium. The discovery of intrinsically disordered proteins (also known as intrinsically unstructured, natively disordered, natively unfolded proteins) has provided new insights and explanations into functionality of many proteins. Intrinsically disordered proteins (IDPs) evolve rapidly and are particularly abundant in eukaryotes. They are associated with signaling, regulation and control pathways. Bioinformatics approaches are now widely used for quick and efficient identification of such proteins even at the proteome levels. This chapter highlights evolutionary trends and functional significance of IDPs, along with the methods to study intrinsically disordered regions.

Intrinsically disordered proteins (IDPs) are enriched in signaling and regulatory functions because disordered segments permit interaction with several proteins and hence the reuse of the same protein in multiple pathways. Understanding IDP regulation is important because altered expression of IDPs is associated with many diseases. Recent studies show that IDPs are tightly regulated and that dosage-sensitive genes encode proteins with disordered segments. The tight regulation of IDPs may contribute to signaling fidelity by ensuring that IDPs are available in appropriate amounts and not present longer than needed. The altered availability of IDPs may result in sequestration of proteins through non-functional interactions involving disordered segments (i.e., molecular titration), thereby causing an imbalance in signaling pathways. We discuss the regulation of IDPs, address implications for signaling, disease and drug development, and outline directions for future research.

ZAP-70 is a tyrosine kinase that functions as a key intermediary in the tyrosine phosphorylation cascade triggered by the activated T cell receptor. Using two SH2 domains arranged in tandem, ZAP-70 detects upstream phosphorylation signals and propagates them to downstream protein targets. Here we review the mechanism of ZAP-70 activation and regulation, with an emphasis on recent structural studies.

In virtually all cases, protein function is governed by the protein's ability to adopt multiple conformations. Ligand binding events are a highly studied class of dynamic molecular adaptation. In a number of systems the adaptation is not driven simply by the ligand binding, but also by interactions with other molecules or ions. This type of allosteric modulation of protein structure, therefore, directly influences its function. Here we describe the development of synthetic antibodies from phage display libraries that are selected to specifically recognize different conformations of the same protein. We focus on maltose binding protein, as a model protein, which undergoes a large conformational change upon binding to its ligand, maltose. Using conformation-specific synthetic antibodies, the affinity of the protein was modulated by dynamically altering the equilibrium between the two conformations in vitro and in vivo. Further, the antibodies were used to rescue the function of a mutant with a drastically lower affinity. The implications for taking into account the conformational dynamics of proteins in designing affinity reagents expands their utility as biotechnology tools and will aid in the development of more sophisticated drugs that can target mechanisms of conformational regulation.

The association of σ factors with the RNA polymerase dictates the expression profile of a bacterial cell. Major changes to the transcription profile are achieved by the use of multiple a factors that confer distinct promoter selectivity to the holoenzyme. The cellular concentration of a σ factor is regulated by diverse mechanisms involving transcription, translation and post-translational events. The number of σ factors varies substantially across bacteria. The diversity in the interactions between σ factors also vary- ranging from collaboration, competition or partial redundancy in some cellular or environmental contexts. These interactions can be rationalized by a mechanistic model referred to as the partitioning of σ space model of bacterial transcription. The structural similarity between different σ/anti-σ complexes despite poor sequence conservation and cellular localization reveals an elegant route to incorporate diverse regulatory mechanisms within a structurally conserved scaffold. These features are described here with a focus on σ/anti-σ complexes from Mycobacterium tuberculosis. In particular, we discuss recent data on the conditional regulation of σ/anti-σ factor interactions. Specific stages of M. tuberculosis infection, such as the latent phase, as well as the remarkable adaptability of this pathogen to diverse environmental conditions can be rationalized by the synchronized action of different a factors.

Enzymes utilizing pyridoxal 5'-phosphate dependent mechanism for catalysis are observed in all cellular forms of living organisms. PLP-dependent enzymes catalyze a wide variety of reactions involving amino acid substrates and their analogs. Structurally, these ubiquitous enzymes have been classified into four major fold types. We have carried out investigations on the structure and function of fold type I enzymes serine hydroxymethy1 transferase and acetylornithine amino transferase, fold type II enzymes catabolic threonine deaminase, D-serine deaminase, D-cysteine desulfhydrase and diaminopropionate ammonia lyase. This review summarizes the major findings of investigations on fold type II enzymes in the context of similar studies on other PLP-dependent enzymes. Fold type II enzymes participate in pathways of both degradation and synthesis of amino acids. Polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity are briefly described. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance.

Despite the presence of many and varied functional groups among the 20 amino acids commonly used in proteins, covalent bonds between these groups are rare. Only disulfide bonds, between pairs of Cys residues arc relatively common. Nevertheless, protein structure analyses continue to reveal novel covalent linkages that result from intramolecular reactions in proteins. Here we describe one such example in which isopeptide bonds are formed between the side chains of lysine and asparagine (or aspartic acid) in an autocatalytic process during protein folding. Bonds of this kind were first discovered in the course of structural analyses of the component proteins of the pili expressed by Streptococcus pyogenes. Isopeptide bonds are now known to be widely present in other pili and in cell-surface adhesins where they provide resistance to physical and chemical stress and may be an evolutionary alternative to disulfide bonds.

Polyketides and non-ribosomal peptides constitute a major class of pharmaceutically important secondary metabolites with diverse biological functions. They are biosynthesized by polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) family of megasynthases. These enzymes can generate enormous diversity in the chemical structures of secondary metabolites by combinatorial use of a limited number of catalytic domains and subtle variations of amino acids in the active site pockets of these enzymatic domains. In this review, we discuss recently developed structural bioinformatics approaches which help in correlating sequence and structural features of the PKS and NRPS megasynthases to the chemical structures of their secondary metabolites biosynthetic products.

Currently, access to rare biomolecular events on long time scales is limited to hundreds of microseconds using conventional molecular dynamics (MD), but generally simulations are run for only a few hundred nanoseconds due to the computational resources needed for these simulations. In order to access relevant events occurring on long time sales, milliseconds and beyond, using conventional hardware, enhanced sampling methods are commonly used. Accelerated molecular dynamics (aMD) is increasingly used in biomolecular simulation because it efficiently allows access to events on long timescales without increasing the number of simulation steps required. Here we review the aMD method and discuss the achievements and limitations of this enhanced sampling simulation technique.

Porphobilinogen deaminase catalyses the formation of 1-hydroxymethylbilane through a stepwise polymerisation of four units of porphobilinogen using a unique dipyrromethane cofactor as a primer. Structural and biochemical studies have suggested residues with catalytic importance, but their specific role in the mechanism and the dynamic behaviour of the protein remains unknown. Dynamics of the protein through three stages of chain elongation were studied using MD simulations to understand the concomitant structural changes. Observations suggest that domain 1 and domain 2 move apart while the cofactor turn region (240-243) moves into the active site and is inclined towards domain 2, thus creating space for the pyrrole moieties added at each stage. Results also suggest possible role of D50, K55 and R149 in active site loop modulation.

Mcl-1 and A1 constitute a subclass within the group of anti-apoptotic Bcl-2 proteins and are shown to be overexpressed in several human cancers. Diverse BH3 domains of different pro-apoptotic Bcl-2 proteins bind to the hydrophobic groove of both these helical bundle proteins with different affinities. Development of any inhibitors for Mcl-1 and A1 requires the knowledge of the behavior of the binding region. In this study, we have carried out molecular dynamics simulations of apo-Mcl-1 and holo-Al systems for a period of 100 ns and compared the results with our earlier studies on Bcl-X_L, another anti-apoptotic protein from a different subclass. During the course of the simulation, the BH3-containing helix H2 is destabilized in Mcl-1, a behavior observed in Bcl-X_L also. The unwinding is attributed to the presence of glycine residues in the segment containing H2. Additionally, this region also contains eight basic residues including three doublets. We have hypothesized that the dibasic motifs could be the cleavage sites for the enzymes that will generate smaller isoforms of Mcl-1 and the unwinding of H2 can aid in the exposure of these cleavage sites. As far as Al is concerned, the BH3-containing helix H2 is mostly stable except the last two helical turns. However, it adopts a completely different orientation within the first 10 ns. This change in orientation realigns the loop region that links H2 and the next helix so that most of the exposed hydrophobic residues can be shielded. The charged residues in both the proteins that are projected towards the hydrophobic groove in the experimentally determined structures are exposed to the solvent during the simulations. This is due to the destabilization of helical regions in which they are present. The unwinding of helix H2 in Mcl-1 and the change in orientation of the same helix in Al point to the flexible nature of hydrophobic groove and this could be important for the binding of diverse BH3 peptides.

Proteins are not classical (Debye) solids. They are deformable polymers and they belong to class of objects known as ‘complex systems’. Fractal dimension based measures have been found to be an extremely useful tool to quantify various aspects of distributions of protein biophysical and biochemical properties. The present work shows that it is possible to separately quantify the self-similar symmetries in mass, hydrophobicity and polarizability distributions and in spatial correlation patterns amongst peptide-dipole units, charged residues, hydrophobic residues, residues with the π-electron cloud, etc. Current review presents multitudes of results to underscore the effectiveness of fractal measures for protein interior and exterior properties.

We have developed a method, called the MOLS method (for mutually orthogonal Latin squares) to exhaustively explore the conformational space of small peptides. Here we used this method to delineate the inherent structure (IS) landscape of the pentapeptide Met-Enkephalin at and above ‘room temperature’. Inherent structures have been defined as structures located at the minima of the energy landscape. From the IS landscape we extracted the probability density and calculated the density of states. To compare the results, we have also sampled the conformations for the peptide through molecular dynamics simulations and periodic rapid quenching. The probability density distributions followed a similar pattern in both methods.