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M Prabhakaran

Publications and source records attributed to M Prabhakaran.

At least 19 recordsLinked to original sources
PubMed2006

Molecular modeling studies of surface decoration of hemoglobin by maleimide PEG.

Surface decoration of hemoglobin (Hb) with six copies of PEG-5K employing thiolation mediated PEGylation platform neutralizes the vasoaconstricive activity of acellular Hb. The molecular size homogeneity of hexaPEGylated Hb, in spite of the fact that the PEGylation is distributed at multiple sites and PEGylation at each of the sites is not quantitative, is an unusual aspect of this PEGylation reaction. We have introduced three cys residues-Cys-13 (alpha), Cys-111 (alpha), and Cys-13 (beta)-onto Hb by molecular modeling. This new mutant Hb with four reactive Cys residues has been used to build molecular models of PEGylated Hbs with two, four, six, and eight PEG-chains of different masses. The calculated loss of surface area was used to design and gain insight into the structure and the surface shielding of PEGylated Hbs. The modeling shows the adequate surface coverage of the protein hemoglobin with six copies of PEG-5K chains and also exhibits more surface coverage of the hemoglobin as compared to that afforded by two copies of PEG-20K chains.

Computer Simulation↗
PubMed2006

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist.

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.

Amino Acid Sequence↗
PubMed2001

Activation of the low oxygen affinity-inducing potential of the Asn108(beta)-->Lys mutation of Hb-Presbyterian on intramolecular alpha alpha-fumaryl cross-bridging.

The Asn108 beta-->Lys mutation in hemoglobin (HbPresbyterian mutation) endows a low O(2) affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two alpha 99 lysine residues also induces a low O(2) affinity into HbA. We have now engineered an alpha alpha-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O(2) affinity-inducing structural perturbations. Despite the presence of the additional epsilon-amino group of Lys108(beta) within the central cavity, the epsilon-amino group of Lys99(alpha alpha) of deoxy Hb-Presbyterian retained high selectivity for alpha alpha-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The alpha alpha-fumaryl cross-linking of Hb-Presbyterian reduced its O(2) affinity much more significantly than that observed with HbA, indicating a synergy between the two low O(2) affinity-inducing structural perturbations. Apparently, the alpha alpha-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O(2) affinity-inducing potential of Lys108(beta) that is generally activated in the presence of chloride. The synergy between the Asn108(beta)-->Lys mutation and the alpha alpha-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, alpha alpha-fumaryl Hb-Presbyterian accessed a low O(2) affinity T-state that is accessed by HbA, alpha alpha-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the alpha alpha-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(alpha) and Lys108(beta) [presumably His103(alpha) and/or Glu101(beta)] to compensate for the extra positive charge of Lys108(beta). Molecular modeling studies identified two potential chloride binding sites per alpha beta dimer within the middle of the central cavity of alphaalpha-fumaryl HbA involving residues His103(alpha), Arg104(beta) and Asn108(beta). The affinity of these sites is increased in alpha alpha-fumaryl Hb-Presbyterian as a result of the Asn108(beta)-->Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the alpha alpha-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the alpha alpha- fumaryl-Hb Presbyterian to access a low O(2) affinity T-state structure much more readily than either Hb-Presbyterian or alpha alpha-fumaryl HbA.

Allosteric Regulation↗
PubMed2000

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.

Allosteric Regulation↗
PubMed2000

Sequencing and model structure of a Naja naja atra protein fragment.

We report the amino acid sequence of a basic protein isolated from the snake venom of Naja naja atra. An automated Edman sequencer was used to determine the 65-residue sequence, aided by electrospray ionization/mass spectrometry. Online reduction and pyridylethylation of the peptide was performed to identify the cysteine residues. Trypsin, chymotrypsin and aspartic digestions were carried out to derive peptide fragments for further sequencing. Fragmented peptides were overlapped to obtain the complete sequence. Molecular mass measurements of the whole protein and its fragments were used as a countercheck for sequence assignment. Further confirmation of the sequence was indicated by sequence homology to other snake venom neurotoxins. A molecular model of the tertiary structure was constructed based on sequence homology, and was refined by global minimization and extensive quality control algorithms. Electrostatic and hydrophobic surface calculations and molecular dynamics simulations were carried out to determine the functional properties of the molecule.

Amino Acid Sequence↗
PubMed1999

Inhibition of beta(S)-chain dependent polymerization by synergistic complementation of contact site perturbations of alpha-chain: application of semisynthetic chimeric alpha-chains.

Mouse alpha(1-30)-horse alpha(31-141) chimeric alpha-chain, a semisynthetic super-inhibitory alpha-chain, inhibits beta(S)-chain dependent polymerization better than both parent alpha-chains. Although contact site sequence differences are absent in the alpha(1-30) region of the chimeric chain, the four sequence differences of the region alpha(17-22) could induce perturbations of the side chains at alpha(16), alpha(20) and alpha(23), the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse alpha(31-141) probably results in the super-inhibitory activity of the chimeric alpha-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20(alpha)-->Gln], a contact site sequence difference, engineered into human-horse chimeric alpha-chain has been investigated to map such a synergistic complementation. Gln20(alpha) has little effect on the O(2) affinity of HbS, but in human-horse chimeric alpha-chain it reduces the O(2) affinity slightly. In the chimeric alpha-chain, Gln20(alpha) increased sensitivity of the betabeta cleft for the DPG influence, reflecting a cross-talk between the alpha(1)beta(1) interface and betabeta cleft in this semisynthetic chimeric HbS. In the human alpha-chain frame, the polymerization inhibitory activity of Gln20(alpha) is higher compared with horse alpha(1-30), but lower than mouse alpha(1-30). Gln20(alpha) synergistically complements the inhibitory propensity of horse alpha(31-141). However, the inhibitory activity of LM-horse chimeric alpha-chain is still lower than that of mouse-horse chimeric alpha-chain. Therefore, perturbation of multiple contact sites in the alpha(1-30) region of the mouse-horse chimeric alpha-chain and its linkage with the inhibitory propensity of horse alpha(31-141) has been now invoked to explain the super-inhibitory activity of the chimeric alpha-chain. The 'linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into alpha-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.

Anemia, Sickle Cell↗
PubMed1997

A new spatial representation of amino acid residues in globular proteins.

For the globular proteins with known three-dimensional structures, an ellipsoid model of each protein was constructed with least volume and its dimensions were derived. The spatial arrangements were made for the C alpha and side chain atoms of that protein within that ellipsoid. This new spatial representation shows the residue position from the centroid, as well as the depth from the surface. The average spatial parameters were then calculated. The correlations between these new spatial parameters and the existing parameters of the amino acid residues were then derived.

Amino Acid Sequence↗
PubMed1996

Internal packing conditions and fluctuations of amino acid residues in globular proteins.

In order to investigate the environmental conditions of amino acid residues in protein molecules, four kinds of packing studies (atomic, geometric, hydrophobic and hydration) were formulated and tested on two proteins; bovine pancreatic trypsin inhibitor (BPTI) and bovine pancreatic ribonuclease S (RNase S). The inter-relationship of these packings on the fluctuations of amino acid residues was analysed by comparing the packing results with the dynamical studies, such as the root-mean-square-deviation values of atomic displacements obtained from the trajectories of molecular dynamics simulation, temperature factor information from crystal structures and residue fluctuations in proteins from continuum model. These analyses yield information about the most fluctuating and most stabilizing residue sites. Comparison of the results obtained by these methods indicate a good agreement, specifying an inverse correlation between the residue packing and fluctuations. This kind of study is helpful in identifying the specific residue sites such as nucleation, receptor binding and antigenic determining sites which in a way indirectly correlates with the functional residues in protein molecules.

Amino Acids↗
PubMed1994

[D-TRP32]neuropeptide Y: a competitive antagonist of NPY in rat hypothalamus.

Neuropeptide Y (NPY) is a potent orexigenic peptide. Structure-activity studies have revealed that nearly the entire sequence of NPY is required to elicit feeding responses. Therefore, in order to develop antagonistic peptides for NPY-induced feeding, we synthesized full-length analogs of NPY, substituting D-Trp in the C-terminal receptor binding region, and screened their activity in rat hypothalamus. Although [D-Trp36]NPY and [D-Trp34]NPY inhibited isoproterenol-stimulated hypothalamic membrane adenylate cyclase activity, [D-Trp32]NPY exhibited no intrinsic activity. Furthermore, [D-Trp32]NPY inhibited [125I]NPY binding to rat hypothalamic membranes with a potency comparable to that of NPY. The presence of 30 and 300 nM concentrations of [D-Trp32]NPY shifted the inhibitory dose-response curve of NPY on isoproterenol-stimulated hypothalamic membrane adenylate cyclase activity parallel to the right with comparable KB values. Moreover, in vivo experiments in rats revealed that [D-Trp32]NPY (10 micrograms) significantly attenuated the 1-h feeding response induced by NPY (1 microgram). Several other substitutions at position 32 including 2-D-Nal resulted in agonist activity, suggesting that there are strict structural requirements to induce the antagonistic property in NPY. These findings confirm that [D-Trp32]NPY is a competitive antagonist of NPY in both in vitro and in vivo systems. Analogs based on [D-Trp32]NPY may have potential clinical application, since NPY has been implicated in the pathophysiology of a number of feeding disorders including obesity, anorexia, and bulimia.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Identification of disulfide bridges in a cardiotoxic peptide by electrospray ionization.

A new method was developed for subjecting a peptide to a specified number of Edman degradation cycles on an automated polypeptide sequencer and desorbing the residual peptide for further investigations. The procedure was applied in combination with electrospray ionization mass spectrometry to identify the four disulfide bridges present in a small tightly bound peptide. The task was accomplished using only a few nanomoles of the intact peptide.

Amino Acid Sequence↗
PubMed1993

Molecular dynamics of sickle and normal hemoglobins.

Molecular dynamics (MD) simulations have been carried out for 62.5 ps on crystal structures of deoxy sickle cell hemoglobin (HbS) and normal deoxy hemoglobin (HbA) using the CHARMM MD algorithm, with a time step of 0.001 ps. In the trajectory analysis of the 12.5-62.5 (50 ps) simulation, oscillations of the radius of gyration and solvent-accessible surface area were calculated. HbS exhibited a general contraction during the simulation, while HbA exhibited a nearly constant size. The average deviations of simulated structures from the starting structures were found to be 1.8 A for HbA and 2.3 A for HbS. The average rms amplitudes of atomic motions (atomic flexibility) were about 0.7 A HbA and about 1.0 A for HbS. The amplitudes of backbone motion correlate well with temperature factors derived from x-ray crystallography. A comparison of flexibility between the alpha- and beta-chains in both HbA and HbS indicates that the beta-chains generally exhibited greater flexibility than the alpha-chains, and that the HbS beta-chains exhibit greater flexibility in the N-terminal and D- and F-helix regions than do those of HbA. The average amplitude of backbone torsional oscillations was about 9 degrees, a value comparable with that of other simulations, with enhanced torsional oscillation occurring primarily at the ends of helices or in loop regions between helices. Comparison of atomic flexibility and torsional oscillation results suggests that the increased beta-chain flexibility results from relatively concerted motions of secondary structure elements. The increased flexibility may play an important role in HbS polymerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Simulation↗
PubMed1992

Location of potential binding sites on deoxy hemoglobin for the design of antigelling agents.

The binding sites of indole-based gelation inhibitors with sickle cell hemoglobin were investigated by two parallel theoretical approaches. A geometric approach originated by Kuntz and co-workers uses a spatial buildup scheme to locate potential binding regions, while a hybrid grid/geometric search method searches for specific indole ring binding pockets over the hemoglobin surface. The binding sites derived from these calculations were tested for their ability to accommodate indole rings by means of accessibility calculations with probes of various radii. These sites were further scanned for van der Waals' overlap and electrostatic interactions. A full 5BrTrp residue was built in each indole ring binding site, and its conformational energy of association with sickle hemoglobin was calculated at that site. Our theoretical results predict a total of 14 potential binding regions, including all of the sites observed from X-ray crystallography, and sites that are consistent with solution nuclear magnetic resonance studies.

Antisickling Agents↗
PubMed1991

Conformational space of alpha 1-4 glycosidic linkage: a molecular dynamics study.

Molecular Dynamics simulations have been carried out for 100 ps on crystal structure of beta-cyclodextrin in vacuo and with explicit inclusion of solvent at constant pressure and constant temperature using the GROMOS MD algorithm, with a time step of 0.005 ps. The conformational space of the glycosidic linkage was studied by calculating two virtual dihedrals connecting the successive glucose units for the 2000 structures saved during the two simulations. Three preferred regions for alpha 1-4 glycosidic linkage were found in both the simulations. The use of these virtual dihedral angles in representing the glycosidic linkage is also brought out from these studies.

Algorithms↗
PubMed1990

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans.

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.

Computer Graphics↗
PubMed1990

The distribution of physical, chemical and conformational properties in signal and nascent peptides.

Signal peptides play a major role in an as-yet-undefined way in the translocation of proteins across membranes. The sequential arrangement of the chemical, physical and conformational properties of the signal and nascent amino acid sequences of the translocated proteins has been compiled and analysed in the present study. The sequence data of 126 signal peptides of length between 18 and 21 residues form the basis of this study. The statistical distribution of the following properties was studied hydrophobicity, Mr, bulkiness, chromatographic index and preference for adopting alpha-helical, beta-sheet and turn structures. The contribution of each property to the sequence arrangement was derived. A hydrophobic core sequence was found in all signal peptides investigated. The structural arrangement of the cleavage site was also clearly revealed by this study. Most of the physical properties of the individual sequences correlated (correlation coefficient approximately 0.4) very well with the average distribution. The preferred occupancy of amino acid residues in the signal and nascent sequences was also calculated and correlated with their property distribution. The periodic behaviour of the signal and nascent chains was revealed by calculating their hydrophobic moments for various repetitive conformations. A graphical analysis of average hydrophobic moments versus average hydrophobicity of peptides revealed the transmembrane characteristics of signal peptides and globular characteristics of the nascent peptides.

Amino Acid Sequence↗
PubMed1990

Secondary structure prediction for the spectrin 106-amino acid segment, and a proposed model for tertiary structure.

A collective secondary structure prediction for the human erythrocyte spectrin 106-residue repeat segment is developed, based on the sequences of nine segments that have been reported in the literature, utilizing a consensus of several secondary structure prediction methods for locating turn regions. The analysis predicts a five-fold structure, with three alpha-helices and two beta-strand regions, and differs from previous models on the lengths of the helices and the existence of beta-strand structure. We also demonstrate that this structural motif can be folded into tertiary structures that satisfy the experimental spectrin data and several general principles of protein organization.

Circular Dichroism↗