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Biomedical subjects

G Govil

Publications and source records attributed to G Govil.

At least 19 recordsLinked to original sources
PubMed2001

NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba Histolytica.

We present the three-dimensional (3D) solution structure of a calcium-binding protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoebiasis affecting millions worldwide. EhCaBP is a 14.7 kDa (134 residues) monomeric protein thought to play a role in the pathogenesis of amoebiasis. The 3D structure of Ca(2+)-bound EhCaBP has been derived using multidimensional nuclear magnetic resonance (NMR) spectroscopic techniques. The study reveals the presence of two globular domains connected by a flexible linker region spanning 8 amino acid residues. Each domain consists of a pair of helix-loop-helix motifs similar to the canonical EF-hand motif of calcium-binding proteins. EhCaBP binds to four Ca(2+) with high affinity (two in each domain), and it is structurally related to calmodulin (CaM) and troponin C (TnC) despite its low sequence homology ( approximately 29%) with these proteins. NMR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. However, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These findings are supported by backbone (15)N relaxation rate measurements and deuterium exchange studies, which reveal low structural order parameters for residues in the central linker region. Earlier, biochemical studies showed that EhCaBP is involved in a novel signal transduction mechanism, distinct from CaM. A possible reason for such a functional diversity is revealed by a detailed comparison of the 3D structure of EhCaBP with that of CaM and TnC. The studies indicate a more open C-terminal domain for EhCaBP with larger water exposed total hydrophobic surface area as compared to CaM and TnC. Further dissimilarities between the structures include the presence of two Gly residues (G63 and G67) in the central linker region of EhCaBP, which seem to impart it a greater flexibility compared to CaM and TnC and also play crucial role in its biological function. Thus, unlike in CaM and TnC, wherein the length and/or composition of the central linker have been found to be crucial for their function, in EhCaBP, both flexibility as well as amino acid composition is required for the function of the protein.

Amino Acid Sequence↗
PubMed2000

A tracked approach for automated NMR assignments in proteins (TATAPRO).

A novel automated approach for the sequence specific NMR assignments of 1HN, 13Calpha, 13Cbeta, 13C'/1Halpha and 15N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate 1HN and 15N chemical shifts with those of 13Calpha, 13Cbeta and 13C'/1Halpha. The information derived from such correlations is used to create a 'master-_list' consisting of all possible sets of 1HN(i), 15N(i)13Calpha(i),13Cbeta(i) 13C'beta(i)/1Halpha(i), 13Calpha(i-1), 13Cbeta(i-1) and 13C'(i-1)/1Halpha(i-1) chemical shifts. On the basis of an extensive statistical analysis of 13Calpha and 13Cbeta chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the master_list and also to translate the protein primary sequence into an array called pps_array. The program then uses the master_list to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so. each assigned residue is tracked in an array called assig_array, with the two-digit code assigned earlier. The assig_array is then mapped onto the pps_array for sequence specific resonance assignment. The program has been tested using experimental data on a calcium binding protein from Entamoeba histolytica (Eh-CaBP, 15 kDa) having substantial internal sequence homology and using published data on four other proteins in the molecular weight range of 18-42 kDa. In all the cases, nearly complete sequence specific resonance assignments (> 95%) are obtained. Furthermore, the reliability of the program has been tested by deleting sets of chemical shifts randomly from the master_list created for the test proteins.

Algorithms↗
PubMed2000

Protective effect of L-arginine against lipid peroxidation in goat epididymal spermatozoa.

L-arginine plays an important role in physiology of spermatozoa and is shown to enhance the metabolism of these cells. We report here the effect of L-arginine on membrane lipid peroxidation of goat epididymal spermatozoa. Both natural peroxidation as well as that induced by UV radiation, freezing and oxidizing agents have been studied. Irrespective of the nature of induction of peroxidation, L-arginine reduces the extent of lipid peroxidation in a concentration dependent manner. Both L-arginine and alpha-tocopherol act synergistically in protecting against lipid peroxidation induced by the above methods. Thus, in order to provide protection against lipid peroxidation, L-arginine may be added in media used to preserve spermatozoa.

Animals↗
PubMed1999

Secondary structure of a calcium binding protein (CaBP) from Entamoeba histolytica.

A calcium binding protein from Entamoeba histolytica, (EhCaBP, M(r) approximately 15 kDa) is the causative agent for amoebiosis and has a very low sequence homology (approximately 30%) with other known CaBPs. Almost complete sequence specific resonance assignments for (1)H, (13)C and (15)N spins in EhCaBP were obtained using double and triple resonance NMR experiments. Qualitative interpretation of the nuclear Overhauser enhancements, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structure of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix-loop-helix motifs. Though this protein has a very small sequence homology with calmodulins, the topological arrangement of the alpha-helices and beta-strands in EhCaBP resemble them.

Amino Acid Sequence↗
PubMed1999

Identification of low-molecular-weight compounds in goat epididymis using multinuclear nuclear magnetic resonance.

Multinuclear nuclear magnetic resonance (NMR) (1H, 13C, and 31P) studies have been performed on aqueous solutions of lyophilysates of cell-free extract, epididymal fluid, and intact cells from caput and cauda regions of epididymis of sacrificed goats. Identification of low-molecular-weight compounds present in different maturation phases of spermatozoa has been carried out. Several low-molecular-weight compounds have been identified by assigning 600 MHz 1H NMR spectra with the help of two-dimensional homonuclear and heteronuclear correlation spectroscopy such as double quantum filtered correlation spectroscopy and heteronuclear single quantum correlation spectroscopy. Homonuclear coupling constants have also been used to get unambiguous assignments of resonances. NMR data were compared with those of standard samples measured at same pH and with those reported in the literature. Identification of several amino acids, carbohydrates, and lipids have been made and their presence has been discussed in relation to their relevance to sperm functions. The presence of beta-alanine and hypotaurine has been reported for the first time in goat epididymis.

Animals↗
PubMed1999

Arginine acts as a protective and reversal agent against glycolytic inhibitors in spermatozoa.

It is known that the amino acid arginine stimulates sperm motility and glycolytic activity. We have earlier studied its efficacy as a stimulator of glycolysis in goat spermatozoa under anaerobic conditions. Here, we have assessed the influence of arginine in reversing the impairment caused by glycolytic inhibitors, iodoacetamide and iodoacetic acid. Glycolysis has been monitored by measuring the consumption of 13C labeled glucose and the amount of 13C labeled lactate produced under different experimental conditions, using 13C NMR. It is observed that both L- and D-arginine are able to prevent and reverse the inhibitory action of glycolytic inhibitors. The reversal effect of arginine gives rise to about eight times higher metabolic activity as compared to the inhibited cells while structurally related amino acids such as nitro-arginine, homo-arginine, lysine and ornithine are ineffective. The energetics of spermatozoa as measured by 31P NMR show a reduction in ATP level in cells incubated with iodoacetamide. Treatment of these cells with both L- and D-arginine restores the ATP level. The results may have significance in the treatment of male infertility.

Animals↗
PubMed1998

A novel palindromic triple-stranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG.

We have carried out NMR and molecular mechanics studies on a complex formed when a palindromic homopyrimidine dodecamer (d-CTTCTCCTCTTC) and a homopurine hexamer (d-GAAGAG) are mixed in 1:1 molar ratio in aqueous solutions. Such studies unequivocally establish that two strands of each oligomer combine to form a triple-stranded DNA structure with a palindromic symmetry and with six T.A:T and six C+. G:C hydrogen-bonded base triads. The two purine strands are placed head to head, with their 3' ends facing each other in the center of the structure. One-half of each pyrimidine strand contains protonated and the other half contains non-protonated cytosines. The two half segments containing protonated cytosines are hydrogen bonded to each of the two purine hexamers through Hoogsteen T.A and C+.G base pairing. The segments containing non-protonated cytosines are involved in Watson-Crick (A:T and G:C) base pairing. This leads to a palindromic triplex with a C2-dyad symmetry with respect to the center of the structure. The complex is less stable at neutral pH, but the cytosines involved in Hoogsteen base pairing remain protonated even under these conditions. Molecular mechanics calculations using NMR constraints have provided a detailed three-dimensional structure of the complex. The entire stretches of purine, and the pyrimidine nucleotides have a conformation close to B-DNA.

Base Composition↗
PubMed1998

Arginine activates glycolysis of goat epididymal spermatozoa: an NMR study.

The present study explores the mechanism underlying the action of L-arginine on the metabolic activity of spermatozoa. Goat epididymal spermatozoa were incubated with different concentrations of L-arginine to determine its effect on the utilization of glucose, fructose, and pyruvate. NMR techniques have been applied to elucidate the effect of L-arginine, L-lysine, and L-ornithine on the glycolysis of epididymal goat spermatozoa. Whereas 31P NMR has been used to estimate the change of pH in the presence of different concentrations of L-arginine, 13C NMR has been used to estimate the substrate consumption and lactate production. At optimal concentration of L-arginine, the forward metabolic rates have been found to increase by two to three times over control experiments. Arginine is not consumed in these reactions, but acts as an activator. Longitudinal relaxation time (T1) measurements indicate that the guanidino group of L-arginine plays an active role in binding to cells. The amino acid L-lysine is less effective, and L-ornithine is ineffective.

Animals↗
PubMed1998

Molecular mechanics calculations on a triple stranded DNA involving C+.G-T and T.A+-C mismatched base triples.

We have carried out molecular modeling of a triple stranded pyrimidine(Y). purine(R): pyrimidine(Y) (where ':' refers to Watson-Crick and '.' to Hoogsteen bonding) DNA, formed by a homopurine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC). Molecular mechanics calculations using NMR constraints have provided a detailed three dimensional structure of the triplex. The entire stretches of purine and the pyrimidine nucleotides have a conformation close to B-DNA. The three strands are held by the canonical C+.G:C and T.A:T hydrogen bonds. The structure also contains two mismatch C+.G-T and T.A+-C base triples which have been characterized for the first time. In the A+-C base-pair of the T.A+-C triple, both hydrogen donors are situated on the purine (A+(1N) and A+(6N)). We observe a unique hydrogen bonding interaction scheme in case of C+.G-T where one acceptor, G(60), is bonded to three donors (C+(3NH), C+(4NH2) and T(3NH)). Though the C+.G-T base triple is less stable than C+.G:C, it is significantly more stable than T.A:T. On the other hand, T.A+-C is as stable as the T.A:T base triad.

Base Pairing↗
PubMed1997

Homopurine and homopyrimidine strands complementary in parallel orientation form an antiparallel duplex at neutral pH with A-C, G-T, and T-C mismatched base pairs.

DNA sequences d-TGAGGAAAGAAGGT (a 14-mer) and d-CTCCTTTCTTCC (a 12-mer) are complementary in parallel orientation forming either Donohue (reverse Watson-Crick) base pairing at neutral pH or Hoogsteen base pairing at slightly acidic pH. The structure of the complex formed by dissolving the two strands in equimolar ratio in water has been investigated by nmr. At neutral pH, the system forms an ordered antiparallel duplex with five A : T and four G : C Watson-Crick base pairs and three mismatches, namely G-T, A-C, and T-C. The nuclear Overhauser effect cross-peak pattern suggests an overall B-DNA conformation with major structural perturbations near the mismatches. The duplex has a low melting point and dissociates directly into single strands with a broad melting profile. The hydrogen-bonding schemes in the mismatched base pairs have been investigated. It has been shown earlier that in acidic pH, the system prefers a triple-stranded structure with two pyrimidine strands and one purine strand. One of the pyrimidine strands has protonated cytosines, forms Hoogsteen base pairing, and is aligned parallel to the purine strand; the other has nonprotonated cytosines and has base-pairing scheme similar to the one discussed in this paper. The parallel duplex is therefore less stable than either the antiparallel duplex or the triplex, in spite of its perfect complementarity.

Base Composition↗
PubMed1997

NMR structural characterisation of a hairpin DNA with two-base loop: solution conformation of d-GGTACIAGTACC.

An inosine-adenosine mismatched base-pair oligonucleotide, d-GGTACIAGTACC has been studied by 1H and 31P NMR spectroscopy. Almost complete 1H and 31P resonance assignments of the oligomer at 0.90 mM concentration and 310 K have been achieved. NMR results demonstrate that the oligomer adopts a hairpin conformation, which has a structure with two purines I6 and A7 forming a two-base loop on a B-DNA stem. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop stacks partially with I6. All the bases are in anti conformation with respect to their respective sugar moiety.

Base Composition↗
PubMed1995

Evidence for A+(anti)-G(syn) mismatched base-pairing in d-GGTAAGCGTACC.

Two-dimensional NMR spectroscopy has been used to study the structure and hydrogen bonding scheme of A:G mismatched base pairing in d-GGTAAGCGTACC at pH 5.8. Under the conditions of our study, the molecule forms a B-DNA helix, with the mismatched bases in the A+ anti)-G(syn) conformation. The adenosine exists in the protonated form. The NOESY spectrum in 90% H2O + 10% 2H2O has been used to assign all observable imino and amino protons including those involved in the A+(anti)-G(syn) base pair. Both the proton donors in the A:G mismatched inter-base hydrogen bonding are situated on adenosine.

Base Composition↗
PubMed1995

NMR characterisation of a triple stranded complex formed by homo-purine and homo-pyrimidine DNA strands at 1:1 molar ratio and acidic pH.

Homo-purine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC) oligomers have been designed such that they are complementary in parallel orientation. When mixed in a 1:1 molar ratio, the system adopts an antiparallel duplex at neutral pH with three mismatched base pairs. On lowering the pH below 5.5, a new complex is formed. The NMR results show the coexistence of a intermolecular pyrimidine.purine:pyrimidine DNA triplex and a single stranded oligopurine at this pH. The triplex is stabilized by five T.A:T, four C+.G:C and two mismatched triads, namely, C+.G-T and T.A-C. This triplex is further stabilized by a Hoogsteen C+.G base-pair on one end. Temperature dependence of the imino proton resonances reveals that the triplex dissociates directly into single strands around 55 degrees C, without duplex intermediates. Parallel duplexes are not formed under any of the conditions employed in this study.

Base Sequence↗
PubMed1994

Interaction of daunomycin with deoxydinucleotide d-CpG by two-dimensional proton magnetic resonance techniques.

Interaction of daunomycin with d-CpG has been studied by taking 500-MHz proton NMR spectra of a mixture of 5.5 mM daunomycin with 11.0 mM d-CpG in the temperature range 277-320 K. The 2D COSY and NOESY spectra of the complex are investigated at 297 K. The nonexchangeable base protons of d-CpG and the ring protons of drug chromophore shift upfield up to approximately 0.27 ppm on interaction. Changes in chemical shift decrease with temperature and are attributed to stacking of drug chromophore between CG and GC base pairs. The sugar is predominantly in the S conformational state for both cytosine and guanine residues in the right-handed helix of the d-CpG complex. The glycosyl angles are about -120 +/- 20 degrees and -90 +/- 20 degrees for C and G residues, respectively. In daunomycin, the spin-spin couplings--J(1'H-2'eqH), J(1'H-2'axH), J(4'H-5'CH3), J(7H-8axH), and J(7H-8eqH)--are altered on complexation with d-CpG. Further, the interproton distances--1'H-2'axH, 7H-8axH, 7H-8eqH, 5'H-8axH, and 5'H-8eqH--are altered significantly on complexation. The observed intermolecular NOEs-CH6-2H, 1H; CH6, CH5-4OCH3; CH1'-2'axH; and GH4'-9COCH3--demonstrate the existence of specific conformation of the complex which reflects the anti-cancerous action of the drug.

DNA↗
PubMed1993

Structural and dynamic membrane properties of alpha-tocopherol and alpha-tocotrienol: implication to the molecular mechanism of their antioxidant potency.

d-alpha-Tocopherol and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but different hydrocarbon "tails". alpha-Tocotrienol has been shown to be more potent in protecting against free radical-induced oxidative stress than alpha-tocopherol. Simple models of phospholipid membrane systems were used to investigate the mechanism of the antioxidant potency of alpha-tocotrienol in terms of its effects on membrane order and reorientation dynamics. Chemiluminescence and fluorescence measurements demonstrated that alpha-tocotrienol exhibits significantly greater peroxyl radical scavenging potency than alpha-tocopherol in phosphatidylcholine liposomes, whereas both antioxidants have identical activity in hexane. This suggests that the antioxidant potency of alpha-tocotrienol requires the membrane environment. When alpha-tocopherol and alpha-tocotrienol were examined for their effects on phospholipid molecular order using conventional ESR spin labeling with 5- and 16-position-labeled doxylstearic acid, although both vitamin E constituents disordered the gel phase and stabilized the liquid-crystalline phase, no differences were observed between the effects of the two compounds. A slightly greater increase (19% vs 15%) in ordering of the liquid-crystalline state due to alpha-tocopherol, however, was discerned in noninvasive 2H NMR experiments. The difference is most noticeable near C10-C13 positions of the phospholipid chain, possibly suggesting alpha-tocotrienol is located closer to the membrane surface. Saturation-transfer ESR, furthermore, revealed that on the time scale tau c = 10(-7)-10(-3) s the rates of rotation about the long molecular axis and of the wobbling motion of the axis are modified to differing extents by the two forms of the vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidants↗
PubMed1993

Solution structure of GCCAAT recognition motif by 2D NMR, spectral simulation, molecular modeling, and distance geometry calculations.

Solution conformation of a self-complementary 14-mer DNA duplex (d-GGATTGGCCAATCC) containing the GCCAAT recognition motif of several transcription factors has been investigated by NMR spectroscopy. Complete resonance assignment of all the protons (except H5',H5'' protons) has been obtained following standard procedures based on two-dimensional NMR techniques. Three-bond coupling constants have been determined by spectral simulation procedures. New strategies have been described and employed for quantifying NOE intensities from the structural point of view. Approximate ranges of gamma torsion angles have been obtained from a selective NOESY experiment, by estimating the J(4'-5'), J(4'-5''), or their sum in the H1'-H4' cross peaks of the spectrum. Likewise, ranges of epsilon torsion angles have been obtained by monitoring the H3' multiplicities in the H8/H6-H3' cross peaks in selective NOESY spectra. With the help of such a total of 73 coupling constraints, 79 NOE intensity constraints, and 108 H-bond constraints, model building has been carried out to obtain a structure which satisfies the constraints. Starting from such a structure, an expanded distance constraint set has been created which has been used for the distance geometry calculations using the program TANDY. In the best structure thus derived, interesting irregularities similar to a BI-BII transition have been observed in the center. The molecule exhibits a bend. The overall base stacking is different from that in either B- or A-DNA models. The base pairs are tilted with respect to the local helix axes. The observed structural features are likely to have important implications for the recognition mechanism of the GCCAAT motif.

Algorithms↗
PubMed1993

Spectroscopic and microscopic studies of buffalo-bull (Bubalus bubalis) spermatozoa.

We have examined the epididymal (caput, corpus and cauda) and ejaculated spermatozoa of bufallo-bull (Bubalus bubalis) employing microscopic and spectroscopic techniques. Fluorescein isothiocyanate conjugated lectins namely concanavalin A (Con A), Dolichos biflorus (DBA), Maclura pomifera (MPA), peanut agglutinin (PNA), soybean agglutinin (SBA) and wheat germ agglutinin (WGA) were used to study the changes in the sperm surface carbohydrate make up as the spermatozoa mature. Quantitative analysis of the lectin binding was made flow cytometrically. 31P-NMR (nuclear magnetic resonance) spectra of the sperms obtained from different regions (head, body and tail) of the epididymis and of the ejaculate were analyzed to assess their metabolic activity. And the kinetics of spin label reduction of these samples was monitored with ESR (electron spin resonance) spectroscopy. These observations are supplemented with the electron microscopic (SEM and TEM) examination of the epididymal and ejaculated spermatozoa.

Animals↗