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P Balaram

Publications and source records attributed to P Balaram.

At least 73 records · Page 4Linked to original sources
PubMed1998

Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals.

An analysis on the nature of alpha-helix stop signals has been carried out, using a dataset of 1057 helices identified from 250 high resolution (</=2.0 A), non-homologous, protein crystal structures. The backbone dihedral angles (phi, psi) of the terminating residue (T) were found to cluster either in the left-handed helical region (alphaL: phi=20 degrees to 125 degrees and psi=-45 degrees to 90 degrees ; 469 helices (44%)) or in the extended region (E: phi=-180 degrees to -30 degrees and psi=60 degrees to 180 degrees and -180 degrees to -150 degrees ; 459 helices (43%)) of the Ramachandran map. These two broad categories of helix stop signals, alphaL and E-terminated helices, were further examined for sequence preferences. Gly residues were found to have an overwhelming preference to occur as the "alphaL-terminator (T)" resulting in the classical Schellman motif, with a strong preference for hydrophobic residues at position T-4 and T+1. In the case of E-terminated helices His, Asn, Leu and Phe were found to occur with high propensity at position T. Quite remarkably Pro residues, with single exception, were absent at position T, but had the highest propensity at position T+1. Examination of the frequencies of hydrophobic (h) and polar (p) residues at positions flanking Gly/Pro permitted delineation of exclusive patterns and predictive rules for Gly-terminated helices and Pro-terminated helices. The analysis reveals that Pro residues flanked by polar amino acids have a very strong tendency to terminate helices. Examination of a segment ranging from T-4 to T+3 appeared to be necessary to determine whether helix termination or continuation occur at Gly residues. The two types of helix termination (alphaL, E) signals also differed dramatically in their solvent accessibility. Gly and Pro residues at helix termini appeared to be strongly conserved in homologous sequences.

Forecasting↗
PubMed1998

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.

Acetone↗
PubMed1998

Purification of an allergenic protein fraction from wheat grain.

An allergenic protein fraction was isolated from wheat gluten by ion exchange and gel filtration chromatography. On sodium dodecyl sulphate gel electrophoresis the molecular mass of the protein was found to be 65 kDa. By Western blotting it was confirmed that the 65 kDa protein was the major allergenic fraction causing dermatitis herpetiformis (DH) and it contained glutamic acid and proline as the major amino acids.

Allergens↗
PubMed1998

Proliferating cell nuclear antigen (PCNA) expression in gestational trophoblastic diseases (GTD).

Gestational Trophoblastic Disease is an abnormal condition of the placenta, the incidence of which is very high in the state of Kerala, India. The proliferative rate of molar placentas in comparison with the normal placentas of comparable gestational age group was done in order to find out its role in the prognosis of this tumor by assessing the expression of PCNA in trophoblasts. PCNA expression was evaluated in 149 trophoblastic tumors and 96 normal placental tissue. The percentage of positive cells was significantly increased in molar placentas of the 1st trimester in comparison to the normal placentas. Correlation of the staining score to the regression pattern of the tumor showed a significant increase in the chemotherapy group when compared to the spontaneously regressing group. But no correlation was found between the percentage of PCNA positive cells with histological grade of the tumor proliferation.

Female↗
PubMed1997

Characterization of a 22-residue peptide derived from a designed ion channel.

We have designed a four-helix protein that is expected to tetramerize in the membrane to form an ion channel with a structurally well-defined pore. This should serve as a model system to study the structural requirements of voltage-sensitive, ion-selective transmembrane channels. We have synthesized the peptide corresponding to the channel-lining helix. Circular dichroism (CD) spectroscopy shows that this peptide is helical in the membrane. Fluorescence resonance energy transfer (FRET) shows that this peptide, at low concentrations, forms aggregates in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes and facilitates ion transport across liposomal membranes. Our data indicate that a component of the designed four-helix protein, i.e., the channel-lining helix, behaves as per design.

Alamethicin↗
PubMed1997

Triosephosphate isomerase from Plasmodium falciparum: the crystal structure provides insights into antimalarial drug design.

BACKGROUND: Malaria caused by the parasite Plasmodium falciparum is a major public health concern. The parasite lacks a functional tricarboxylic acid cycle, making glycolysis its sole energy source. Although parasite enzymes have been considered as potential antimalarial drug targets, little is known about their structural biology. Here we report the crystal structure of triosephosphate isomerase (TIM) from P. falciparum at 2.2 A resolution. RESULTS: The crystal structure of P. falciparum TIM (PfTIM), expressed in Escherichia coli, was determined by the molecular replacement method using the structure of trypanosomal TIM as the starting model. Comparison of the PfTIM structure with other TIM structures, particularly human TIM, revealed several differences. In most TIMs the residue at position 183 is a glutamate but in PfTIM it is a leucine. This leucine residue is completely exposed and together with the surrounding positively charged patch, may be responsible for binding TIM to the erythrocyte membrane. Another interesting feature is the occurrence of a cysteine residue at the dimer interface of PfTIM (Cys13), in contrast to human TIM where this residue is a methionine. Finally, residue 96 of human TIM (Ser96), which occurs near the active site, has been replaced by phenylalanine in PfTIM. CONCLUSIONS: Although the human and Plasmodium enzymes share 42% amino acid sequence identity, several key differences suggest that PfTIM may turn out to be a potential drug target. We have identified a region which may be responsible for binding PfTIM to cytoskeletal elements or the band 3 protein of erythrocytes; attachment to the erythrocyte membrane may subsequently lead to the extracellular exposure of parts of the protein. This feature may be important in view of a recent report that patients suffering from P. falciparum malaria mount an antibody response to TIM leading to prolonged hemolysis. A second approach to drug design may be provided by the mutation of the largely conserved residue (Ser96) to phenylalanine in PfTIM. This difference may be of importance in designing specific active-site inhibitors against the enzyme. Finally, specific inhibition of PfTIM subunit assembly might be possible by targeting Cys13 at the dimer interface. The crystal structure of PfTIM provides a framework for new therapeutic leads.

Amino Acid Sequence↗
PubMed1997

Effects of organic solvents on protein structures: observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide.

A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (< 10%) have little effect on the overall folded conformation of lysozyme as seen from 1H NMR chemical shift dispersion. At increasing DMSO concentrations (> 10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by approximately 50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain-side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel beta-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of non-aqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins.

Animals↗
PubMed1997

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

Acetone↗
PubMed1997

Expression of epidermal growth factor receptor in gestational trophoblastic diseases.

Gestational trophoblastic disease is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of this disease is vague and methods to determine the malignant potential are limited. Placentae of normal (50) and molar pregnancies (95), including 32 showing persistent disease, were used for the study. Epidermal growth factor (EGFR), expression was evaluated by immunohistochemistry using monoclonal antibody, and quantified using the 125I-EGF-binding assay. EGFR was expressed more strongly in molar placentae than in normal placentae of similar gestational age, and was strong in the molar placentae of all gestational ages, whereas expression was mainly restricted to the first and second trimesters in normal placentae. Moles with early presenting symptoms (before 16 weeks) were at a higher risk of developing persistent disease. To conclude, the immunohistochemical study shows high expression of EGFR in early normal placentae and in moles, linking its role to the proliferative and differentiating activity of trophoblasts. Tumours with a histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGFR. The present observations also suggest the possibility of mutated EGF receptors being present in persistent trophoblastic disease.

Adult↗
PubMed1997

Chemoprevention of oral leukoplakia with vitamin A and beta carotene: an assessment.

We conducted a double-blind placebo-controlled trial to evaluate the chemopreventive potential of either vitamin A alone or beta carotene alone in subjects with oral leukoplakia in Kerala, India. We randomised 160 fishermen and women with oral precancerous lesions to receive oral vitamin A (retinyl acetate 300,000 IU/week x 12 months, n = 50), or beta carotene (360 mg/week x 12 months, n = 55), or placebo (n = 55). Blood, saliva and urine samples were collected at baseline and at exit to study serum micronutrients and mutagenicity assays. Biopsies of the mucosal lesions at entry were performed for histopathological exclusion of malignancy. The subjects were examined once every 2 months to establish clinical response of lesions and toxicity, if any. The results are based on 43 complaint subjects on placebo, 42 on vitamin A and 46 on beta carotene. The complete regression rates were: 10% in the placebo arm, 52% with vitamin A and 33% with beta carotene (P < 0.0001). Homogeneous leukoplakias and smaller lesions responded better than non-homogeneous and larger lesions. No major toxicities were observed. Half of the responders with beta carotene and two thirds with vitamin A relapsed after stopping supplementation. Serum beta carotene concentration increased substantially with beta carotene administration while with vitamin A supplementation there was no change in serum retinol levels. In the vitamin A treated group there was a significant decrease in serum alpha tocopherol. Vitamin A administration resulted in a significant remission of oral leukoplakia without any side effects of prolonged vitamin A supplementation. The results of this study, as well as those from previous studies, appear to provide strong supporting evidence to justify long term trials with vitamin A in subjects with high-risk leukoplakias with oral cancer as an endpoint.

Adult↗
PubMed1997

Design of two-helix motifs in peptides: crystal structure of a system of linked helices of opposite chirality and a model helix-linker peptide.

BACKGROUND: An attempt is being made to produce two-helix bundles that are soluble in apolar media, without the use of a rigid template. The approach relies on the use of stereochemically constrained amino acids for helix construction, while a flexible linker is obtained by the use of an epsilon-aminocaproic acid residue (Acp). The Acp linker has appropriate NH and COOH termini to connect to the N and C termini of the helices, a flexible (CH2)5 moiety and sufficient length to make the desired assembly. RESULTS: The conformations in crystals (determined by X-ray diffraction analyses) are described for a partial assembly consisting of a 7-residue helix with Acp (helix-Acp) and for two assemblies of 7-residue helices with Acp (helix-Acp-helix) in which the chiralities of the helices are L,L (already published) and L,D (this publication). The Acp linker is extended away from the helix in the L,L analog in a zig-zag manner, but assumes a helical conformation in the L,D analog. The two helices in the L,L and L,D analogs are displaced laterally by the linker, but in neither case has the linker folded the molecule into the desired U-conformation. Cell parameters for Boc-L-Val-L-Ala-L-Leu-Aib-L-Val-L-Ala-L-Leu-Acp-D-Val-D -Ala-D-Leu-Aib-D-Val-D-Ala-D-Leu-OMe are space group P4(1) with a = b = 10.094(6) A and c = 93.383(12) A. CONCLUSIONS: Strong hydrogen bonds (NH...O=C) between the displaced helices of one molecule and the displaced helices of a neighboring molecule, which form near the linker of each helix-linker-helix assembly, appear to dominate in both the L,L and L,D crystal. The (CH2)5 segment of the linker readily adopts different conformations that result in the L and D helices packing in a similar spatial motif. Greater conformational control at the linking segment or introduction of specific interhelix interactions may be necessary in order to achieve U-type folding between neighboring helices in a single molecule.

Crystallography, X-Ray↗
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↗
PubMed1997

Hydrogen bonding in peptide helices. Analysis of two independent helices in the crystal structure of a peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe.

The crystal structure determination of the heptapeptide Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe reveals two peptide helices in the asymmetric unit. Crystal parameters are: space group P2(1), a = 10.356(2) A, b = 19.488(5) A, c = 23.756(6) A, beta = 102.25(2) degrees, V = 4685.4 A3, Z = 4 and R = 5.7% for 7615 reflections [I > 3 sigma (I)]. Both molecules adopt largely alpha-helical conformations with variations at the C-terminus. Helix type is determined by analysing both 4-->1 and 5-->1 hydrogen-bond interactions and comparison with the results of analysis of protein structures. The presence of two 4-->1 hydrogen-bond interactions, besides four 5-->1 interactions in both the conformations provides an opportunity to characterize bifurcated hydrogen bonds at high resolution. Comparison of the two helical conformations with related peptide structures suggests that distortions at the C-terminus are more facile than at the N-terminus.

Amino Acid Sequence↗
PubMed1997

Expression of epidermal growth factor in gestational trophoblastic disease (GTD).

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Adolescent↗
PubMed1997

Metabolic enzymes as potential drug targets in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria that is fatal in many cases. Emergence of drug resistant strains of P. falciparum requires that new drug targets be identified. This review considers in detail enzymes of the glycolytic pathway, purine salvage pathway, pyrimidine biosynthesis and proteases involved in catabolism of haemoglobin. Structural features of P. falciparum triosephosphate isomerase which could be exploited for parasite specific drug development have been highlighted. Utility of P. falciparum hypoxanthine-guanine-phosphoribosyltransferase, adenylosuccinate synthase, dihydroorotate dehydrogenase, thymidylate synthase-dihydrofolate reductase, cysteine and aspartic proteases have been elaborated in detail. The review also briefly touches upon other potential targets in P. falciparum.

Animals↗
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

Mutations and polymorphisms in the p53, p21 and p16 genes in oral carcinomas of Indian betel quid chewers.

India has one of the world's highest incidences of oral cancer. It is believed that the widespread habit of betel quid chewing is an important risk factor as it exposes the oral mucosa to known carcinogens. It also induces physical abrasions, which may create mitogenic environments during wound healing as gateways for infections. A recent study from our laboratories identified human papillomavirus (HPV) DNA, mostly of the high-risk types HPV-16 and HPV-18, in 67 of 91 oral cancer lesions from a cohort of Indian patients consisting mostly of betel quid users. This suggested a viral etiology of some lesions but tumorigenesis in the absence of viruses in other lesions. Here, we examined whether the p53 gene, whose function is abrogated by the product of the HPV gene E6, would be mutated in those oral cancers that were free of HPV DNA, and we found point mutations at known hot spots for mutational alteration of p53 in 4 of 23 lesions. We also considered the possibility that p21, a target of regulation by the p53 protein, may be mutationally altered in tumors with a functional p53 gene. While we did not identify mutations in the p21 gene, 6 of 11 lesions contained a polymorphism that may be associated with cancer. Interestingly, 3 of 23 lesions had mutations in the p16 gene, a third regulator of the cell cycle which is frequently mutated in melanoma but rarely in other cancers, with 1 lesion even having a mutation in the p53 as well as in the p16 gene. Our data point to p53 and p16 as gene targets of oral carcinogenesis, with chemicals in the betel quid possibly functioning in these tumors as carcinogens.

Areca↗