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P Balaram

Publications and source records attributed to P Balaram.

At least 91 records · Page 5Linked to original sources
PubMed1996

A designed beta-hairpin peptide in crystals.

Beta-hairpin structures have been crystallographically characterized only in very short acyclic peptides, in contrast to helices. The structure of the designed beta-hairpin, t-butoxycarbonyl-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe in crystals is described. The two independent molecules of the octapeptide fold into almost ideal beta-hairpin conformations with the central D-Pro-Gly segment adopting a Type II' beta-turn conformation. The definitive characterization of a beta-hairpin has implications for de novo peptide and protein design, particularly for the development of three- and four-stranded beta-sheets.

Amino Acid Sequence↗
PubMed1996

Covalent reinforcement of a fragile region in the dimeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding.

Urea and guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme, and engineered interface mutants have been monitored by circular dichroism, fluorescence, and size-exclusion chromatography. Equilibrium unfolding studies show biphasic transitions, with a plateau between 3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energy transfer employing an (aminoethylamino) naphthalenesulfonyl (AEDANS) label at the active site residue, Cys198. AEDANS was also specifically incorporated at position Cys155 in the mutant protein T155C. Direct excitation of this extrinsic fluorophore in the wild type protein (labeled at Cys198) and mutant T155C (labeled at Cys155) showed remarkable differences in the unfolding profiles. C155 AEDANS has a transition centered at 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea). Unfolding studies monitored by following intrinsic fluorescence of Trp residues which are located in a small structural domain suggest that this region of the protein is intrinsically fragile. The stable equilibrium intermediate is identified to be an ensemble of partially unfolded aggregated species by gel filtration studies. The chaotrope-induced denaturation of TS appears to proceed through a partially unfolded intermediate that is stabilized by aggregation. Dissociation and loss of structure occur concomitantly at high denaturant concentrations. Introduction of two symmetrically positioned disulfide bridges across the dimer interface in the triple mutant T155C/E188C/C244T (TSMox) stabilized the protein against denaturant-induced unfolding. Aggregate formation was completely abolished in the mutant TSMox, which also enhanced the overall structural stability of the protein. Structural reinforcement of the fragile interface in thymidylate synthase results in dramatic stabilization toward chaotrope-induced unfolding.

Binding Sites↗
PubMed1996

Solid state and solution conformations of a helical peptide with a central Gly-Gly segment.

The influence of amino acids with contrasting conformational tendencies on the stereochemistry of oligopeptides has been investigated using an octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe, which contains two helix-promoting Aib residues and a central helix-destabilizing Gly-Gly segment. Single crystal x-ray diffraction studies reveal that a 3(10)-helix is formed up to the penultimate Aib residue, at which point there is a helix reversal in the backbone, reminiscent of a C-terminal 6-->1 hydrogen bond. The curious feature in the crystal is the solvation of the possible 6-->1 bond by a CH3OH molecule, where the OH is inserted between O(3) and N(8) and participates in hydrogen bonds with both. The cell parameters are as follows: space group P2(1)2(1)2(1), a = 10.649 (4) A, b = 15.694 (5) A, c = 30.181 (8) A, R = 6.7% for 3427 data (magnitude of F0 > 3 sigma F) observed to 0.9 A. Nuclear magnetic resonance studies in CDCl3 using NH group solvent accessibility and nuclear Overhauser effects as probes are consistent with a 3(10)-helical conformation. In contrast, in (CD3)2SO, unfolding of the central segment results in a multiple beta-turn structure, with beta-turn conformations populated at residues 1-2, 3-4, and 6-7. CD studies in methanol-2,2,2-trifluoroethanol (TFE) mixtures also provide evidence for a solvent-dependent structural transition. Helical conformations are populated in TFE, while type II beta-turn structures are favored in methanol.

Chemical Phenomena↗
PubMed1996

Peptide design: crystal structure of a helical peptide module attached to a potentially nonhelical amino terminal segment.

The peptide Boc-Gly-Dpg-Gly-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe has been designed to examine the structural consequences of placing a short segment with a low helix propensity at the amino terminus of a helical heptapeptide module. The Gly-Dpg-Gly segment is a potential connecting element in the synthetic construction of a helix-linker-helix motif. Crystal parameters for the peptide are P21, a = 8.651 (3) A, b = 46.826 (13) A, c = 16.245 A, beta = 90.13 (3) degrees, Z = 4; 2 independent molecules/asymmetric unit. The structure reveals almost identical conformations for the two independent molecules. The backbone is completely helical for residues 2-9, with on 4 --> 1 hydrogen bond and six 5 --> 1 hydrogen bonds. The alpha, alpha-di-n-propylglycine residue adopts a helica conformation. Gly (1) adopts an extended conformation resulting in a nonhelical N-terminus, with the Boc group swinging away from the helix. the lateral association of helices in the beta axis direction is unusual in that the helix axes are directed up or down (parallel or antiparallel) by pairs: decrease decrease increase increase decrease decrease, etc.

Amino Acid Sequence↗
PubMed1996

Omega amino acids in peptide design: incorporation into helices.

Incorporation of easily available achiral omega-amino acid residues into an oligopeptide results in substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic. The central Gly-Gly segment of the helical octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe(1) has been replaced by delta-amino-valeric acid (delta-Ava) residue in the newly designed peptide Boc-Leu-Aib-Val-delta-Ava-Leu-Aib-Val-OMe(2). 1H-nmr results clearly suggest that in the apolar solvent CDCl3, the delta-Ava residue is accommodated into a folded helical conformation, stabilized by successive hydrogen bonds involving the NH groups of Val(3), delta-Ava(4), and Leu(5). The delta-Ava residue must adopt a gauche-gauche-trans-gauche-gauche conformation along the central polymethylene unit of the aliphatic segment, a feature seen in an energy-minimized model conformation based on nmr parameters. The absence of hydrogen bonding functionalities, however, limits the elongation of the helix. In fact, in CDCl3, the folded conformation consists of an N-terminal helix spanning residues 1-4, followed by a Type II beta-turn at residues 5 and 6, whereas in strongly solvating media like (CD3)2SO, the unfolding of the N-terminal helix results in beta-turn conformations at Leu(1)-Aib(2). The Type II beta-turn at the Leu(5)-Aib(6) segment remains intact even in (CD3)2SO.CD comparisons of peptides 1 and 2 reveal a "nonhelical" spectrum for 2 in 2,2,2-trifluoroethanol.

Amino Acids↗
PubMed1996

Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase.

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.

Bacterial Proteins↗
PubMed1996

Ultrastructural analysis of the adjacent epithelium of oral squamous cell carcinoma.

Fifteen biopsies of the immediate adjacent epithelium of oral squamous cell carcinoma were examined under light and electron microscopy. Light microscopic examination of one micron thick sections revealed that the majority of lesions (67%) had hyperplastic or mildly dysplastic epithelium while the remaining (33%) had moderate to severe dysplasia. Ultrastructural observations showed that all these lesions had subcellular alterations similar to those seen in frank malignant oral tissue, particularly in the lower half of the epithelium. Important ultrastructural changes observed included bizarre nuclei of basal and lower spinal cells, enlarged and multiple nucleoli, presence of interchromatin and perichromatin granules, loss of desmosomes and marked spongiosis as well as disturbed cellular maturation sequences in the keratinocytes evidenced by abnormal and irregular distribution of maturation markers such as keratohyalin granules and tonofilaments. The present study thus shows the value of electron microscopy in the detection of malignant changes in the adjacent epithelium of oral squamous cell carcinoma.

Carcinoma, Squamous Cell↗
PubMed1996

Observation of a mixed antiparallel and parallel beta-sheet motif in the crystal structure of Boc-Ala-Ile-Aib-OMe.

A diastereomeric mixture of the tripeptide Boc-Ala-Ile-Aib-OMe crystallized in the space group P1 from CH3OH/H2O. The unit cell parameters are a = 10.593(2) A, b = 14.377(3) A, c = 17.872(4) A, alpha = 104.41(2) degrees, beta = 90.55(2) degrees, gamma = 106.91(2) degrees, V = 2512.4 A3, Z = 4. X-Ray crystallographic studies show the presence of four molecules in the asymmetric unit consisting of two pairs of diastereomeric peptides, Boc-L-Ala-L-Ile-Aib-OMe and Boc-L-Ala-D-Ile-Aib-OMe. The four molecules in the asymmetric unit form a rarely found mixed antiparallel and parallel beta-sheet hydrogen bond motif. The Ala and (L,D)-Ile residues in all the four molecules adopt the extended conformations, while the phi, psi values of the Aib residues are in the right-handed helical region. In one of the molecules the Ile sidechain adopts the unusual gauche conformation about the C beta-C gamma bond.

Crystallization↗
PubMed1996

Conformation-activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2.

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating 1-aminocycloalkane-1-carboxylic acid (Ac(n)c) and alpha,alpha-dialkylglycines (Deg, diethylglycine; Dpg, n,n-dipropylglycine and Dbg, n,n-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx=Ac(7)c, I; Ac(8)c, II; Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded beta-turn conformations in CDCl3 and (CD3)2SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C5) conformations in solution. Peptides I-V exhibit high activity in inducing beta-glucosaminidase release from rabbit neutrophils, with ED50 values ranging from 1.4-8.0 x 10(-11) M. In human neutrophils the Dxg peptides III-V have ED50 values ranging from 2.3 x 10(-8) to 5.9 x 10(-10) M, with the activity order being V > IV > III. While peptides I-IV are less active than the parent, For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors.

Amino Acid Sequence↗
PubMed1996

A model for the interaction of trifluoroethanol with peptides and proteins.

The structural stabilizing property of 2,2,2-trifluoroethanol (TFE) in peptides has been widely demonstrated. More recently, TFE has been shown to enhance secondary structure content in globular proteins, and to influence quaternary interactions in protein multimers. The molecular mechanisms by which TFE exerts its influence on peptide and protein structures remain poorly understood. The present analysis integrates the known physical properties of TFE with a variety of experimental observations on the interaction of TFE with peptides and proteins and on the properties of fluorocarbons. Two features of TFE, namely the hydrophobicity of the trifluoromethyl group and the hydrogen bonding character (strong donor and poor acceptor), emerge as the most important factors for rationalising the observed effects of TFE. A model is proposed for TFE interaction with peptides which involves an initial replacement of the hydration shell by fluoroalcohol molecules, a process driven by apolar interactions and favourable entropy of dehydration. Subsequent bifurcated hydrogen-bond formation with peptide carbonyl groups, which leave intramolecular interactions unaffected, promotes secondary structure formations.

Entropy↗
PubMed1996

Crystal structures of a nonapeptide helix containing alpha, alpha-di-n-butylglycine (Dbg), Boc-Gly-Dbg-Ala-Val-Ala-Leu-Aib-Val-Leu-OMe.

Two crystal structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue alpha, alpha-di-n-butylglycyl, reveal a mixed 3(10)-/alpha-helical conformation. Residues 1-7 adopt phi, psi values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has phi, psi values of -40, -37 degrees and -46, -40 degrees in the two crystals with the two butyl side chains mostly extended in each. Peptide molecules in the crystals pack into helical columns. The crystal parameters are: C50H91N9O12, space group P2(1), with a = 9.789(1) A, b = 20.240(2) A, c = 15.998(3) A, beta = 103.27(1); Z = 2, R = 10.3% for 1945 data observed > 3 sigma (F) and C50H91N9O12.3H2O, space group P2(1), with a = 9.747(3) A, b = 21.002(8) A, c = 15.885(6) A, beta = 102.22(3) degrees, Z = 2, R = 13.6% for 2535 data observed > 3 sigma (F). The observation of a helical conformation at Dbg suggests that the higher homologs in the alpha, alpha-dialkylated glycine series also have a tendency to stabilize peptide helices.

Norleucine↗
PubMed1996

Immunology of oral cancer--a review.

Cancer of the oral cavity forms the major cancer in India constituting about 20% of all cancers. In spite of the advances made in diagnostic and therapeutic modalities, the morbidity and mortality due to this disease remains high. The overall five year survival rate is only 34%. The reason for this is mainly lack of markers for early detection, prognosis and identification of the high risk group. Immune depression is recognised as a consistent metabolic defect in oral cancer patients and our study shows that this precedes the development of the malignancy. The cancer probably enhances the depression. Monitoring of the immunoregulatory status has shown to correlate well with the prognosis of the patient in our study. Hence the monitoring of the various immune parameters would probably be a reliable marker to identify a high risk group in precancerous lesions, a prognostic indicator in oral cancer patients and an intermediate marker in intervention studies.

Antibodies, Neoplasm↗
PubMed1995

A designed beta-hairpin peptide.

A synthetic octapeptide, Boc-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe (1) has been designed as a model for a beta-hairpin conformation. Circular dichroism spectra in various organic solvents reveal a single negative band at 214-217 nm consistent with beta-sheet structures. NMR studies in CDCl3 and C6D6 establish the solvent shielded nature of the Leu(1), Val(3), Leu(6) and Val (8) NH groups. Nuclear Overhauser effects are observed between Val(7) C alpha H and Val(2) C alpha H protons providing strong support for a beta-hairpin conformation. Several important diagnostic interresidue NOEs establish a Type II' beta-turn conformation for the D-Pro-Gly segment and extended conformations for the amino and carboxyl terminal tripeptide arms. The high solubility of the beta-hairpin peptide in organic solvents holds promise for the development of models for three and four stranded beta-sheets.

Amino Acid Sequence↗
PubMed1995

Biradicals as ESR probes of conformations in model beta-turn peptides.

Electron spin-spin exchange interaction in biradicals has been examined for its potential usefulness in the conformational analysis of peptides in solution. Three peptides with high propensity to adopt beta-turn conformations in solution, Boc-Cys-Pro-Xxx-Cys-NHMe (Xxx = Leu, 1; Xxx = Aib, 2; Xxx = Tyr, 3), have been modified into biradicals by spin labeling the thiols groups. Analysis of electron spin resonance spectra of these peptides in a variety of solvents and at different temperatures suggests that the population of folded conformations follows the order 2 > 1 > 3.

Amino Acid Sequence↗
PubMed1995

Human papillomaviruses in 91 oral cancers from Indian betel quid chewers--high prevalence and multiplicity of infections.

India has one of the world's highest incidences of oral cancer. The habit of chewing betel quid is widespread and is suspected to play a role in the etiology of this disease. Studies in many other countries have also pointed to a role for human papilloma-viruses (HPVs) in the etiology of some oral cancers. In this study we analyzed biopsies from 91 Indian oral cancer patients, most of whom were betel quid chewers, by PCR amplification and direct DNA sequencing. HPV DNA was detected in 74% of these lesions, of which 41% had multiple HPV infections. Among the lesions from different oral sites, lesions of the tongue had the highest rate (9 of 11) of HPV infection. These HPV prevalences are among the highest ever reported in oral cancers. As to individual HPV types, prevalences of HPV-6, HPV-11, HPV-16 and HPV-18 were 13%, 20%, 42% and 47%, respectively. No additional known or novel HPV types were detected. To understand the unexpectedly high prevalences of the "low-risk" types HPV-6 and HPV-11, we compared the subtypes and variants that were found in oral cancers against those from benign genital warts from the same patient population but found no differences. The high prevalence of HPV in the oral cancers of these Indian patients suggests that viral infection is an important etiological component, with betel quid probably causing additional mutagenic steps in the carcinogenic process.

Areca↗
PubMed1995

Enhancing peptide antigenicity by helix stabilization.

The engineering of antigenic determinants on super secondary structures using de novo design approaches often involves synthesis of long peptide chains (35-80 residues long). This communication illustrates that the stabilization of secondary structure by rational design can also greatly enhance immunogenicity and antigenicity, but in much shorter peptide sequences (21 residues long). A peptide epitope the sequence of which has been derived from the C-terminus of the chicken riboflavin carrier protein (cRCP), H2N-Tyr-His-Ala-Cys-Gln-Lys-Lys-Leu-Leu- Lys-Phe-Glu-Ala-Leu-Gln-Gln-Glu-Glu-Gly-Glu-Glu-OH, has been chosen for analysis. Helical conformations were induced in the peptide in aqueous trifluoroethanol. Analogs were designed to stabilize this conformation in water by either the introduction of appropriately spaced ion pairs or the strongly helix nucleating residue alpha-aminoisobutyric acid (Aib), substituted for Ala/Gly, thus affording a comparison of the helix stabilization strategies. Circular dichroism (CD) results demonstrate that all the designed analogs are appreciably more helical than the parent peptide in 50% aqueous trifluoroethanol. Peptide antisera were raised for all analogs in rabbits. The affinities of these antisera for the native protein antigen, determined using a chaotrope disrupted binding assay, correlated very well with the helix content determined by CD.

Amino Acid Sequence↗
PubMed1995

beta-turn conformations in crystal structures of model peptides containing alpha,alpha-di-n-propylglycine and alpha,alpha-di-n-butylglycine.

The crystal state conformations of three peptides containing the alpha,alpha-dialkylated residues, alpha,alpha-di-n-propylglycine (Dpg) and alpha,alpha-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Ala-OMe (I) and Boc-Ala-Dbg-Ala-OMe (II) adopt distorted type II beta-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: phi = 66.2 degrees, psi = 19.3 degrees; III: phi = 66.5 degrees, psi = 21.1 degrees) deviate appreciably from ideal values for the i + 2 residue in a type II beta-turn. In both peptides the observed (N...O) distances between the Boc CO and Ala (3) NH groups are far too long (I: 3.44 A; III: 3.63 A) for an intramolecular 4-->1 hydrogen bond. Boc-Ala-Dpg-Ala-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules IIA and IIB adopt consecutive beta-turn (type III-III in IIA and type III-I in IIB) or incipient 3(10)-helical structures, stabilized by two intramolecular 4-->1 hydrogen bonds. In all four molecules the bond angle N-C alpha-C' (tau) at the Dxg residues are > or = 110 degrees. The observation of conformational angles in the helical region of phi,psi space at these residues is consistent with theoretical predictions.

Amino Acid Sequence↗