Biomedical subjects

C Ramakrishnan

Publications and source records attributed to C Ramakrishnan.

At least 37 records · Page 2Linked to original sources

PubMed1997

A role for HDJ-2/HSDJ in correcting subnuclear trafficking, transactivation, and transrepression defects of a glucocorticoid receptor zinc finger mutant.

All steroid receptors possess a bipartite nuclear localization signal sequence (NLS) that localizes within the second zinc finger of their DNA-binding domain. Fine-structure mapping of the rat glucocorticoid receptor (rGS) NLS identified a composite signal composed of three distinct proto-NLSs that function effectively when present in unique pairs. At least one of the rGR proto-NLSs appears to influence receptor trafficking within the nucleus, as revealed by a unique nuclear staining pattern of receptors possessing a point mutation (i.e., arginine at position 496; R496), at proto-NLS, pNLS-2. Specifically, carboxyl-terminal-truncated rGRs possessing various point mutations at R496 localized within a limited number of large foci in nuclei of transiently transfected COS-1 cells. R496 mutations did not affect subnuclear targeting when present in full-length rGR, reflecting a protective effect of the receptor's ligand-binding domain that can be exerted in cis and in trans. The effects of rGR R496 mutations on subnuclear targeting were not autonomous because we also observed a coincident localization of hsp70, the 70-kDa heat shock protein, within nuclear foci that include r496 mutant receptors. The elimination of R496 mistargeting by overexpression of an hsp70 partner (i.e., the DnaJ homologue, HDJ-2/HSDJ) suggests that the hsp70/DnaJ chaperone system is mobilized to specific sites within the nucleus in response to inappropriate targeting or folding of specific mutant receptors. HDJ-2/HSDJ overexpression also corrects defective transactivation and transrepression activity of R496 mutant GRs. Thus, molecular chaperones, such as members of the hsp70 and DnaJ families, may survey the nucleus for misfolded proteins and actively participate in their refolding into biologically active conformational states.

Amino Acid Sequence↗

PubMed1997

Beta-hairpins in proteins revisited: lessons for de novo design.

Beta-Hairpins with short connecting loops (1-5 residues) have been identified from a data set of 250 non-homologous, high resolution (< or =2.0 A) protein crystal structures. The conformational preferences of the loop segments have been analyzed with the specific aim of identifying frequently occurring motifs. Type I' and II' beta-turns were found to have a high propensity for occurrence in two residue loops. For three and four residue loops, the major conformational motif in the linking segments is alphaR-alphaR-alphaL (type I beta-turn followed by a residue in a left-handed helical conformation) and alphaR-alphaR-alphaR-alphaL (a pi-turn motif), respectively. The present larger data set confirms the high occurrences of these motifs which have been identified in earlier analyses. In addition to type I' and type II' beta-turns, several examples of type I beta-turn nucleated two residue loop hairpins, in spite of having an opposing sense of twist to that of type I' beta-turn, have also been observed. Examination of these frequently occurring motifs (flanked by extended conformation [beta]) in the data set reveals that the motifs beta-alphaR-alphaR-alphaL-beta and beta-type I'-beta have equal propensity and type II' indeed having highest propensity to nucleate beta-hairpins. The larger number of examples in this study allows the estimation of the specific amino acid preferences for loop positions in two, three and four residue loops. Small polar residues Asn, Asp, Ser, Thr, Gly and Pro in general have a high propensity for the loop positions but they reveal specific positional preferences in these frequently occurring motifs. There are no strong compositional preferences in the strand segments. Amino acid pair correlations across strands also do not show any significant pattern, with the exception of Cys-Cys pairs. Several Cys-Cys pairs have been identified at the non-hydrogen bonded positions of beta-hairpins; as many as six are disulfide bonded pairs. An examination of longer loop length hairpins reveals that the distortions of hairpins nucleated by tight turns (two residues) are much less frequently observed. The results presented in this study provide inputs for the de novo design of consensus loop segments in synthetic hairpins.

Amino Acid Sequence↗

PubMed1996

A procedure for the prediction of temperature-sensitive mutants of a globular protein based solely on the amino acid sequence.

Temperature-sensitive (Ts) mutants of a protein are an extremely powerful tool for studying protein function in vivo and in cell culture. We have devised a method to predict those residues in a protein sequence that, when appropriately mutated, are most likely to give rise to a Ts phenotype. Since substitutions of buried hydrophobic residues often result in significant destabilization of the protein, our method predicts those residues in the sequence that are likely to be buried in the protein structure. We also indicate a set of amino acid substitutions, which should be made to generate a Ts mutant of the protein. This method requires only the protein sequence. No structural information or homologous sequence information is required. This method was applied to a test data set of 30 nonhomologous protein structures from the Protein Data Bank. All of the residues predicted by the method to be > or = 95% buried were, in fact, buried in the protein crystal structure. In contrast, only 50% of all hydrophobic residues in this data set were > or = 95% buried. This method successfully predicts several known Ts and partially active mutants of T4 lysozyme, lambda repressor, gene V protein, and staphylococcal nuclease. This method also correctly predicts residues that form part of the hydrophobic cores of lambda repressor, myoglobin, and cytochrome b562.

Amino Acid Sequence↗

PubMed1996

Disallowed Ramachandran conformations of amino acid residues in protein structures.

An analysis of the nature and distribution of disallowed Ramachandran conformations of amino acid residues observed in high resolution protein crystal structures has been carried out. A data set consisting of 110 high resolution, non-homologous, protein crystal structures from the Brookhaven Protein Data Bank was examined. The data set consisted of a total of 18,708 non-Gly residues, which were characterized on the basis of their backbone dihedral angles (phi, psi). Residues falling outside the defined "broad allowed limits" on the Ramachandran map were chosen and the reported B-factor value of the alpha-carbon atom was used to further select well defined disallowed conformations. The conformations of the selected 66 disallowed residues clustered in distinct regions of the Ramachandran map indicating that specific phi, psi angle distortions are preferred under compulsions imposed by local constraints. The distribution of various amino acid residues in the disallowed residue data set showed a predominance of small polar/charged residues, with bulky hydrophobic residues being infrequent. As a further check, for all the 66 cases non-hydrogen van der Waals short contacts in the protein structures were evaluated and compared with the ideal "Ala-dipeptide" constructed using disallowed dihedral angle (phi, psi) values. The analysis reveals that short contacts are eliminated in most cases by local distortions of bond angles. An analysis of the conformation of the identified disallowed residues in related protein structures reveals instances of conservation of unusual stereochemistry.

Amino Acids↗

PubMed1996

Motifs and conformational analysis of amino acid residues adjoining beta-turns in proteins.

Using a data set of 250 non-homologous high-resolution globular proteins, a systematic analysis of the conformations that precede and succeed (positions i and i + 3) the various classical beta-turn types has been carried out. The collective conformation of a specific beta-turn type, including the flanking positions, termed motif, has been studied. In all the four turn types, the majority of examples are preceded and succeeded by extended conformation. Some of the other observations are: (1) In a type I beta-turn, Gly at position i + 3 has a higher favorability to occur with positive phi and does not prefer the major motif beta-alpha R-alpha R-beta. (2) The left-handed alpha-helical conformation (alpha L) is not preferred at both the flanking positions for type I' and II' beta-turns. (3) The beta--beta motif is favourable for all the turn types and the motif beta--alpha L very highly favourable for type I.

Amino Acids↗

PubMed1995

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120.

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264, 82-85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C alpha torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (alpha L) and that in family 2 is right-handed helical (alpha R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereochemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens.

Amino Acid Sequence↗

PubMed1994

Conformational characteristics of asparaginyl residues in proteins.

Backbone conformations at 1064 asparaginyl residues in 123 non-homologous, high-resolution X-ray structures of proteins were analysed. Asn adopts conformations in left-handed alpha-helical region and other partially allowed regions in the Ramachandran map more readily than any other non-glycyl residue. Asn conformational clusters in the (phi, psi) regions of left-handed alpha-helix right-handed alpha-helix and extended (beta) strands were investigated in detail for their occurrence in various secondary structures, especially in beta-turn regions. Preferences were observed for Asn conformations in different positions in various beta-turn types, including the first and fourth positions of the turn. Asparaginyl residues with extended conformations are found to occur frequently in irregular regions, although they are expected to occur predominantly in extended strands or in the third position of type II beta-turns. Asn conformations at the N-cap positions of helices strongly prefer extended conformation than alpha L, which seems to be characteristic of non-glycyl residues at that position. In the liners connecting two extended strands and those connecting an alpha-helix and an extended strand, Asn with alpha L or alpha R conformation is more favoured than Asn with the beta-conformation. Analysis of Asn-Asn doublets and Asn-X-Asn triplets permitted identification of conformational families in such sequences. Results of this investigation provide useful hints in modelling Asn-rich regions in proteins such as malaria parasite coat protein.

Asparagine↗

PubMed1993

Termination of right handed helices in proteins by residues in left handed helical conformations.

An analysis of 636 helical segments, ranging in length from 4 to 32 residues, from 123 independent protein crystal structures reveals that helix termination by residues in left handed (alpha 1) helical conformations is a common occurrence. Gly and Asn residues are the most frequent alpha L helix terminators, with the former having a very high propensity to adopt such conformations. The alpha R-alpha R-alpha R-alpha L segment at the C termini of protein helices often possesses a 6--> 1 (pi-type) hydrogen bond between the CO of residue i and the NH of residue i + 5 with residue i + 4 occurring in the alpha L conformation. A stereochemical analysis of 216 examples shows that in 62 cases the 6-->1 hydrogen bond is absent. The present analysis provides a quantitative measure of the propensity of the 20 amino acids to adopt alpha L helix terminating conformations.

Hydrogen Bonding↗

PubMed1993

Modelling multiple disulphide loop containing polypeptides by random conformation generation. The test cases of alpha-conotoxin GI and endothelin I.

A general procedure for arriving at 3-D models of disulphide-rich polypeptide systems based on the covalent cross-link constraints has been developed. The procedure, which has been coded as a computer program, RANMOD, assigns a large number of random, permitted backbone conformations to the polypeptide and identifies stereochemically acceptable structures as plausible models based on strainless disulphide bridge modelling. Disulphide bond modelling is performed using the procedure MODIP developed earlier, in connection with the choice of suitable sites where disulphide bonds could be engineered in proteins (Sowdhamini, R., Srinivasan, N., Shoichet, B., Santi, D.V., Ramakrishnan, C. and Balaram, P. (1989) Protein Engng, 3, 95-103). The method RANMOD has been tested on small disulphide loops and the structures compared against preferred backbone conformations derived from an analysis of putative disulphide subdatabase and model calculations. RANMOD has been applied to disulphide-rich peptides and found to give rise to several stereochemically acceptable structures. The results obtained on the modelling of two test cases, alpha-conotoxin GI and endothelin I, are presented. Available NMR data suggest that such small systems exhibit conformational heterogeneity in solution. Hence, this approach for obtaining several distinct models is particularly attractive for the study of conformational excursions.

Amino Acid Sequence↗

PubMed1992

Orthogonal beta beta motifs in proteins.

A super-secondary structural motif comprising two orthogonally oriented beta-strands connected by short linking segments of less than or equal to 5 residues has been identified from a data set of 65 independent protein crystal structures. Of the 42 examples from 14 proteins, a vast majority have only a single residue as the linking element. Analysis of the conformational angles at the junction reveals that the recently described type VIII beta-turn occurs frequently at the connecting hinge, while the type II beta-turn is also fairly common.

Animals↗

PubMed1992

Conformational studies on beta-bend containing a cis peptide unit.

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively and these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds--either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis' and trans' conformations are possible for the prolyl residue as also the C gamma-endo and C gamma-exo puckerings, and (iv) for Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans' conformation at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.

Alanine↗

PubMed1990

Conformations of disulfide bridges in proteins.

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of less than or equal to 2.0 A, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (phi, psi) values at Cys residues, bridge dihedral angles chi ss, chi i1, chi j1, chi i2, and chi j2, the distances C alpha i-C alpha j and C beta i-C beta j between the C alpha and C beta atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson [1981) Adv. Protein Chem. 34, 167-330). Disulfide bridging across antiparallel extended strands is observed in alpha-lytic protease, crambin, and beta-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.

Cystine↗

PubMed1989

Conformational analysis of cyclolinopeptide A, a cyclic nonapeptide: nuclear Overhauser effect and energy minimization studies.

The conformation of cyclolinopeptide A [cyclo(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring cyclic nonapeptide has been investigated in dimethylsulfoxide solution by 270 MHz 1H-nmr. A complete assignment of all C alpha H and NH resonances has been accomplished using two-dimensional correlated spectroscopy and nuclear Overhauser effects (NOEs). Analysis of interresidue NOEs and JHNC alpha H values permit construction of a molecular model for the cyclic peptide backbone. The crude model derived from nmr has been used as a starting point for energy minimization, which yields a refined structure largely compatible with nmr observations. The major features of the conformation of cyclolinopeptide A are a Type VI beta-turn centered at Pro(1)-Pro(2), with a cis peptide bond between these residues and a gamma-turn (C7 structure) centered at Ile(6). Two intramolecular hydrogen bonds Val(9) CO--Phe(3)NH (4----1) and Leu(5) CO--Ile(7)NH (3----1) are observed in the low-energy conformation. The limited solvent accessibility observed for the Val(9) and Leu(5) NH groups in the nmr studies are rationalized in terms of steric shielding.

Magnetic Resonance Spectroscopy↗

PubMed1989

Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis.

A computer modeling procedure for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cystine disulfide crosslinks has been developed. Residue pairs with C alpha-C alpha distances of less than or equal to 6.5 A and C beta-C beta distances of less than or equal to 4.5 A are chosen for geometrical fixation of S atoms using the program MODIP. The stereochemistry of the modeled disulfides is evaluated using limits for the structural parameters of the various torsion angles and S-S bond length in the disulfide bridge. The ability of the procedure to correctly model disulfides has been checked with examples of cystine peptides of known crystal structures and 103 disulfide bridges from 25 available protein crystal structures determined at less than or equal to 2 A resolution. An analysis of results on three proteins with engineered disulfides, T4 lysozyme, dihydrofolate reductase and subtilisin, is presented. Two positions for the introduction of 'stereochemically optimal' disulfides are identified in subtilisin.

Disulfides↗

PubMed1987

Nuclear Overhauser effects as probes of peptide structure. A diagnostic for left-handed helical conformations.

The conformational dependence of the interresidue interproton distances in peptides, C alpha H ... Ni + 1 H and NiH ... Ni + 1 H, have been used to identify zones of sterically allowed phi, psi space, where both distances are less than 3A and expected to yield nuclear Overhauser effects (NOEs). L-residues in left-handed helical conformations are expected to yield both interresidue NOEs and also an appreciable intraresidue NiH----C alpha iH NOE. The effect of cutoff distances has been evaluated. Experimental results on three model peptides illustrate the utility of these NOEs in identifying L-residues at the i + 2 position of Type II and I' beta-turns. Simultaneous observation of both interresidue NOEs may also be indicative of conformational heterogeneity in specific cases, as illustrated for a single residue in a decapeptide.

Magnetic Resonance Spectroscopy↗

PubMed1987

A novel supersecondary structure in globular proteins comprising the collagen-like helix and beta-turn.

A structure consisting of the polyproline-II or collagen-like helix immediately succeeded by a beta-turn is seen in several synthetic peptides and has been suggested to be the conformational requirement for proline hydroxylation in nascent procollagen. Using a simple algorithm for detecting secondary structures, we have analysed crystal structure data on 40 globular proteins and have found eight examples of the collagen-helix + beta-turn supersecondary structure in 15 proteins that contain the collagen-like helical segments.

Algorithms↗

PubMed1987

Conformation of glycyl residues in globular proteins.

Glycine is unique among the amino acids in view of its symmetric nature. While the overall distribution of glycyl residues in the (phi, psi) plane is near-symmetric, there can be certain preferences for the individual conformations. An analysis of the observed glycyl conformations in 70 proteins has been carried out to find the influence of residues adjoining the glycyl residues. For this purpose, the (phi, psi) plane has been divided into two regions: the region in which phi is negative and the region in which phi is positive. The analysis is done in terms of the number of conformations occurring in these regions. It has been found that while the overall percentage distribution of glycyl residues between the positive and the negative phi regions is 54:46, the distribution shows asymmetry when the examples are sorted out in terms of X-Gly and Gly-Y doublets. The asymmetry becomes more prominent when the data are sorted out into triplets X-Gly-Y. Using the available information, it has been possible to designate 25 triplets as P-predominant and 19 as N-predominant. An examination of P-predominant triplets for possible occurrence in beta-bends having one of the conformations in the positive phi region shows that only 25% are of this nature. Thus, the P-preference of P-predominant triplets is not an outcome of the bend formation alone and must be an inherent property of these triplets.

Amino Acid Sequence↗

PubMed1985

Studies on hydrogen bonds. Part V--Hydrogen bonding in energy minimization studies of peptides.

An energy term, representing the N-H...O type of hydrogen bond, which is a function of the hydrogen bond length (R) and angle (theta) has been introduced in an energy minimization program, taking into consideration its interpolation with the non-bonded energy for borderline values of R and theta. The details of the mathematical formulation of the derivatives of the hydrogen bond function as applicable to the energy minimization have been given. The minimization technique has been applied to hydrogen bonded two and three linked peptide units (gamma-turns and beta-turns), and having Gly, Ala and Pro side chains. Some of the conformational highlights of the resulting minimum energy conformations are a) the occurrence of the expected 4----1 hydrogen bond in all of the burn-turn tripeptide sequences and b) the presence of an additional 3----1 hydrogen bond in some of the type I and II tripeptides with the hydrogen bonding scheme in such type I beta-turns occurring in a bifurcated form. These and other conformational features have been discussed in the light of experimental evidence and theoretical predictions of other workers.

Chemical Phenomena↗