Protein and Peptide Letters - Volume 14, Issue 7, 2007

This is the second and concluding part of the special issue of “Protein Peptide Letters” which presents some state-of-the art contributions in multi-dimensional research at the interface of Protein Peptide Informatics and Drug Designing. The first part focused at specific experimental and computational studies on Protein Kinases, GRPs, and peptide based vaccine designs and structuralbinding properties of several drug targets in Plasmodium falciparum, HIV-I and Japanese Encephalitis Virus. This issue presents some novel algorithms, computational techniques, servers, softwares and databases providing wider applications in Structure Based Drug Design (SBDD) and related issues in Genomics and Proteomics. Drug development research in the past decades has focused on the enzyme-binding-pocket model of drug targets. Advances headed towards peptide and gene based rational drug and vaccine designs now require knowledge of the structural biology of the target, co-receptor and inhibitor molecules. The ab-initio method (PEPstr) and corresponding web-server developed by H. Kaur, A. Garg and G. P. S. Raghava for prediction of tertiary structure of small bioactive peptides makes a useful contribution in this context. Prediction and analysis of the binding interactions of the drug with the receptor (target) molecules is crucial in drug designing projects. Computational methods based on surface complementarity and stereochemistry are widely used for this purpose to enable docking of protein - protein and protein - ligands. However, modifications in the existing docking algorithms are often required in deciphering the stochastic interactions that account for diversity and affinity in surface-complementarity. The all-atom based Monte Carlo method and useful application software named ParDOCK developed by Prof. B. Jayaram's research group in the supercomputing lab at IIT Delhi is an important contribution in this regard. The paper by A. Gupta, A. Gandhimathi, P. Sharma and B. Jayaram presents this procedure with validation results and applications. The applications and scope of gene and protein data banks in next generation research and development in Medical Biotechnology and Pharmacology are remarkable. Multiple sequence alignment and homology modeling have no doubt made the major chunk of harvesting the fruits of knowledge from the ever-expanding databases of experimental data on the Internet. However these datamining techniques are not limitation-free to meet the challenges of Nature. The innovative approach of alignment-free classification of protein sequences presented in the paper by S. Deshmukh, S. Khaitan, D. Das, M. Gupta and P. P. Wangikar provides a distinct alternative with significant scope. Dr. Wangikar's research group had recently reported a new method of classifying protein structure using geometrically invariant structural patterns. Extended application of this algorithm together with dynamic programming, as reported in his paper with A. Dalal and S. Deshmukh, provides an efficient method for protein structure alignment. This method is found to reduce the computational time by 30 to 60 times as compared to the execution time of the conventional methods for average sized protein chains. Two useful contributions to protein structure modeling, analysis or comparison of specific structural attributes are made by Dr. Sekar's group, viz - (i) comprehensive data base (CADB-3.0) of conformation angles of known protein structures (ii) advanced web server for online display of Ramchandran Plot of the confirmation angles of the polypeptide chain of a protein with demarcation of secondary structural elements and regions within any given set or range of values of these angels. Applications illustrated in the corresponding papers - (i) by K. Gopalakrishnan, S. S. Sheik, C. Vasuki Ranjani, A. Udayakumar and K. Sekar; and (ii) K. Gopalakrishnan, G. Sowmiya, S. S. Sheik and K. Sekar - include targets of certain drugs. C. Ramakrishnan, B. Lakshmi, A. Kurien, D. Devipriya and Dr. N. Srinivasan have analyzed the presence of conformational angles (,φ) in non-redundant, high resolution crystal structures of proteins and peptides focusing on those ranges of pair of these angles that are disallowed in the Ramchandran plots....

Among secondary structure elements, β-turns are ubiquitous and major feature of bioactive peptides. We analyzed 77 biologically active peptides with length varying from 9 to 20 residues. Out of 77 peptides, 58 peptides were found to contain at least one β-turn. Further, at the residue level, 34.9% of total peptide residues were found to be in β- turns, higher than the number of helical (32.3%) and β-sheet residues (6.9%). So, we utilized the predicted β-turns information to develop an improved method for predicting the three-dimensional (3D) structure of small peptides. In principle, we built four different structural models for each peptide. The first ‘model I’ was built by assigning all the peptide residues an extended conformation ( = Ψ = 180°). Second ‘model II’ was built using the information of regular secondary structures (helices, β-strands and coil) predicted from PSIPRED. In third ‘model III’, secondary structure information including β-turn types predicted from BetaTurns method was used. The fourth ‘model IV’ had main-chain , Ψ angles of model III and side chain angles assigned using standard Dunbrack backbone dependent rotamer library. These models were further refined using AMBER package and the resultant Cα rmsd values were calculated. It was found that adding the β-turns to the regular secondary structures greatly reduces the rmsd values both before and after the energy minimization. Hence, the results indicate that regular and irregular secondary structures, particularly β-turns information can provide valuable and vital information in the tertiary structure prediction of small bioactive peptides. Based on the above study, a web server PEPstr (http://www.imtech.res.in/raghava/pepstr/) was developed for predicting the tertiary structure of small bioactive peptides.

We report here an all-atom energy based Monte Carlo docking procedure tested on a dataset of 226 proteinligand complexes. Average root mean square deviation (RMSD) from crystal conformation was observed to be ∼ 0.53 Å. The correlation coefficient (r2) for the predicted binding free energies calculated using the docked structures against experimental binding affinities was 0.72. The docking protocol is web-enabled as a free software at www.scfbioiitd. res.in/dock.

Protein sequences vary in their length and are not readily amenable to conventional data mining techniques that need mapping in a fixed dimensional space. Thus, majority of the current methods for protein sequence classification are based on alignment of the query sequence either with a sequence or a profile of the sequence family. We present a method for mapping of protein sequences in a fixed dimensional descriptor space. The descriptors such as amino acid content and amino acid pair association rules were used along with routinely available classification methods. An experiment on one hundred Pfam families showed classification accuracy of 98% with support vector machines classifier. Information gain based feature selection helped simplify the model and improve accuracy. Interestingly, a large number of the selected features were based on the association rules of Glycine or Aspartic acid residues suggesting their role in the conserved loops among evolutionarily related proteins. Further, in another experiment, the approach was tested for classification of proteins from 39 Pfam families of protein kinases. Support vector machines classifier provided an accuracy of approximately 96%. The method provides an alternative to conventional profile based methods for protein sequence classification.

Classification of newly determined protein structures is important in understanding their function and mechanism of action. Currently available methods employ a global structure alignment strategy and are computationally expensive. We propose a two-step methodology with a quick screen to significantly reduce the number of candidate structures followed by global structure alignment of the query structure with the reduced set. We represent a protein structure as a sequence of local structures, codified in the form of geometric invariants. Geometric invariants are quantities that remain unchanged under transformations such as translation and rotation. Protein structures represented as multi-attribute sequences are aligned via dynamic programming to identify close neighbors of the query structure. The query structure is then compared with this reduced dataset using conventional structure comparison methods to predict its functional class. For a typical protein structure, the screening method was able to reduce the protein data bank to mere 200 proteins while preserving structurally closest neighbor in the reduced set. This has resulted in 30 to 60 fold improvement in the execution time. We present the results of leave-one-out classification experiment on ASTRAL-95 domains and comparison with SCOP classification hierarchy.

Transitions in amino-acid conformation angles tend to accompany various structural modifications in protein structures. Thus, to benefit the modeling of protein structures, the Conformation Angles DataBase (CADB-3.0) has been updated to visualize the conformational angles in varied regions (fully, generously, additionally and disallowed regions). In addition, options are provided to display the angles in the secondary structural elements (α-helix, β-sheet and 310-helix) of the Ramachandran plot. The database is being updated periodically and can be accessed over the World Wide Web at the following URL: http://cluster.physics.iisc.ernet.in/cadb/.

The Ramachandran plot displays the main chain conformation angles ( and Ψ) of the polypeptide chain of a protein molecule. The paper reports the updated version of the Ramachandran plot web server and has several improved options for displaying the conformation angles in various regions. In addition, options are provided to display the conformation angles in various secondary structural elements and regions within the user specified  and Ψ values in the plot. The updated version is accessible at the following URL: http://dicsoft1.physics.iisc.ernet.in/rp/

Using a data set of 454 crystal structures of peptides and 80 crystal structures of non-homologous proteins solved at ultra high resolution of 1.2 Å or better we have analyzed the occurrence of disallowed Ramachandran (, Ψ) angles. Out of 1492 and 13508 non-glycyl residues in peptides and proteins respectively 12 and 76 residues in the two datasets adopt clearly disallowed combinations of Ramachandran angles. These examples include a number of conformational points which are far away from any of the allowed regions in the Ramachandran map. According to the Ramachandran map a given (, Ψ) combination is considered disallowed when two non-bonded atoms in a system of twolinked peptide units with ideal geometry are prohibitively proximal in space. However, analysis of the disallowed conformations in peptide and protein structures reveals that none of the observations of disallowed conformations in the crystal structures correspond to a short contact between non-bonded atoms. A further analysis of deviations of bond lengths and angles, from the ideal peptide geometry, at the residue positions of disallowed conformations in the crystal structures suggest that individual bond lengths and angles are all within acceptable limits. Thus, it appears that the rare tolerance of disallowed conformations is possible by gentle and acceptable deviations in a number of bond lengths and angles, from ideal geometry, over a series of bonds resulting in a net gross effect of acceptable non-bonded inter-atomic distances.

Lectins are carbohydrate binding proteins with important roles in many biological processes such as adhesion. Here we have identified 11 potential lectins from Mycobacterium tuberculosis H37Rv genome, using a comprehensive bioinformatics analysis, which will provide helpful clues in molecular mapping of pathogenesis and perhaps also serve as potential drug targets.

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A “repeat” corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A “domain” corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.

PrTX-I, a non-catalytic and myotoxic Lys49-PLA2 from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), α-tocopherol and α-tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 Å resolution. All complexes crystals are isomorphous and belong to the space group P21 whereas native PrTX-I crystals belong to the P3121.

Temporin-1Vb, -1Oe, -1DRb, and -1TGb (10-8 - 10-6M) produced significant (p<0.05) and concentrationdependent stimulatory effects on insulin secretion from clonal rat BRIN-BD11 cells without increased release of lactate dehydrogenase. Temporin-1Va and temporin-1Vc (10-8 - 10-6M) also stimulated insulin-release but were cytotoxic at 10-6M. Temporin-1DRa was without effect. The temporins at 10-7 M had no effect on intracellular calcium concentrations suggesting that they stimulate insulin release via a KATP channel- independent pathway.

Leptin plays an important role in the regulation of food intake and thermogenesis, regulates long term energy balance and reproductive function and its concentrations are closely linked to body mass index. Leptin secretion is influenced by many factors and the age-related changes in different hormones might modify circulating leptin concentrations. Sex dimorphism in leptin concentrations has been clearly shown in previous studies and its concentrations were lower in men than in women in all decades of life. Insulin growth factor-I (IGF-I) is a peptide growth factor that is present in all types of physiologic fluids and is also produced by connective tissue cell types and its autocrine/paracrine secretion is nearly always present within tissues. There is a physiological decline of the growth hormone (GH)/IGF-I axis with ageing and in addition, insulin, thyroid hormones and the supply of dietary energy may directly regulate the circulating levels of the IGFs and growth hormone binding protein (GHBP). Furthermore, there is no doubt that GH participates in the regulation of body composition, and with advanced age there is a decrease in muscle and an increase in adiposity associated with a decline in GH and total IGF-I. The biological activities of the IGF ligands are modulated by the family of high affinity GHBP. Sex hormone binding globulin (SHBG) concentrations are thought to be regulated primarily through opposing actions of sex steroids on hepatic SHBG production, with oestrogen stimulating and androgen inhibiting SHBG production, and thyroid hormones are also a potent stimulator of SHBG production concentrations. Some studies support an independent IGFBP3 contribution to SHBG variability and these findings are compatible with the hypothesis that some of the anabolic effects ascribed to the GH/IGF axis may be caused by SHBG-mediated changes in testosterone activity or SHBG/total testosterone index.

We present the observation of separated protein bands after polyacrylamide (PAA) gel electrophoresis based on the staining-free detection of their ultra violet (UV)-induced fluorescence employing deep UV confocal fluorescence microscopy. Mixtures of the three biological compounds β-Galactosidase (from Escherichia coli), apo-Transferrin (bovine) and bovine serum albumin (BSA) have been separated and a staining free detection limit below 80 pg (7.0x108 molecules) per band has been achieved. This corresponds to ∼270 molecules in the detection volume for confocal microscopy.

Free radicals induce protein modifications, often associated to many biological phenomena. This mini-review overviews the approaches we have used to elucidate the radical-induced damages on sulfur-containing enzymes, such as ribonuclease and lysozyme, and the possible post-translational mechanism of the damage to another cell compartment, such as lipid domain.

The process of nucleocytoplasmic exchange of various macromolecules and metabolites between the nuclear and cytoplasmic compartment is crucial for cell function and survival. It is also closely involved with several pathogeneses including cancer and viral infections. Here, we will discuss the current understanding of the classical nuclear import pathway and the factors that are essential for this process.

Purified endoglucanase from C. biazotea mutant 51SMr was successfully immobilized on Eudragit L-100, with 75 % yield of immobilization. This method improved the kinetic and thermodynamic properties of the enzyme. Immobilization significantly decreased entropy and enthalpy of inactivation of biocatalyst and made it functionally and thermodynamically more stable and reusable compared to free one.