PubMed Health⌕ Search

Biomedical subjects

M R Murthy

Publications and source records attributed to M R Murthy.

At least 19 recordsLinked to original sources
PubMed2001

Structural studies on the empty capsids of Physalis mottle virus.

The three-dimensional crystal structure of the empty capsid of Physalis mottle tymovirus has been determined to 3.2 A resolution. The empty capsids crystallized in the space group P1, leading to 60-fold non-crystallographic redundancy. The known structure of Physalis mottle virus was used as a phasing model to initiate the structure determination by real-space electron-density averaging. The main differences between the structures of the native and the empty capsids were in residues 10 to 28 of the A-subunit, residues 1 to 9 of the B-subunit and residues 1 to 5 of the C-subunit, which are ordered only in the native virus particles. An analysis of the subunit disposition reveals that the virus has expanded radially outward by approximately 1.8 A in the empty particles. The A-subunits move in a direction that makes 10 degrees to the icosahedral 5-fold axes of symmetry. The B and C-subunits move along vectors making 12 degrees and 15 degrees to the quasi 6-fold axes. The quaternary organization of the pentameric and hexameric capsomeres are not altered significantly. However, the pentamer-hexamer contacts are reduced. Therefore, encapsidation of RNA appears to cause a reduction in the particle radius concomittant with the ordering of the N-terminal arm in the three subunits. These structural changes in Physalis mottle virus appear to be larger than the corresponding changes observed in viruses for which both the empty and full particle structures have been determined.

Binding Sites↗
PubMed2001

Variability of calcium binding to EF-hand motifs probed by electrospray ionization mass spectrometry.

The modulation of calcium binding by the EF-hand motifs present in a calmodulin (CAM) homologue, a calcium binding protein (CaBP) from Entamoeba histolytica by three external parameters-pH, ligand coordinator EGTA, and fragmentor voltage was investigated by mass spectrometry. Calcium binding follows expected patterns at highly acidic and alkaline pH with the preponderance of the apo and the completely saturated forms, respectively. Surprisingly, additional nonspecific binding is observed near neutral pH. Studies on EGTA chelation and effects of fragmentor voltage showed cooperativity in calcium removal in at least one of the domains. Similar studies on a smaller construct containing the two high affinity carboxy terminal sites revealed interesting differences and provided an estimate of the specificity and tolerance of the EF-hand motifs to calcium binding and removal.

Calcium↗
PubMed2001

Conformation and structural transitions in the EF-hands of calmodulin.

Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.

Amino Acid Sequence↗
PubMed2000

Protein thermal stability: insights from atomic displacement parameters (B values).

The factors contributing to the thermal stability of proteins from thermophilic origins are matters of intense debate and investigation. Thermophilic proteins are thought to possess better packed interiors than their mesophilic counterparts, leading to lesser overall flexibility and a corresponding reduction in surface-to-volume ratio. These observations prompted an analysis of B values reported in high-resolution X-ray crystal structures of mesophilic and thermophilic proteins. In this analysis, the following aspects were addressed: (1) frequency distribution of normalized B values (B' factors) over all the proteins and for individual amino acids; (2) amino acid compositions in high B value regions of polypeptide chains; (3) variation in the B values from core to the surface of proteins in terms of their radius of gyration; and (4) degree of dispersion of normalized B values in spheres around the Calpha atoms. The analysis revealed that (1) Ser and Thr have lesser flexibility in thermophiles than in mesophiles, (2) the proportion of Glu and Lys in high B value regions of thermophiles is higher and that of Ser and Thr is lower and (3) the dispersion of B values within spheres at Calpha atoms is similar in mesophiles and thermophiles. These observations reflect plausible differences in the dynamics of thermophilic and mesophilic proteins and suggest amino acid substitutions that are likely to change thermal stability.

Amino Acids↗
PubMed1999

Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker.

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction.

Amino Acid Sequence↗
PubMed1999

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.

Animals↗
PubMed1999

Three-dimensional structure of physalis mottle virus: implications for the viral assembly.

The structure of the T=3 single stranded RNA tymovirus, physalis mottle virus (PhMV), has been determined to 3.8 A resolution. PhMV crystals belong to the rhombohedral space group R 3, with one icosahedral particle in the unit cell leading to 20-fold non-crystallographic redundancy. Polyalanine coordinates of the related turnip yellow mosaic virus (TYMV) with which PhMV coat protein shares 32 % amino acid sequence identity were used for obtaining the initial phases. Extensive phase refinement by real space molecular replacement density averaging resulted in an electron density map that revealed density for most of the side-chains and for the 17 residues ordered in PhMV, but not seen in TYMV, at the N terminus of the A subunits. The core secondary and tertiary structures of the subunits have a topology consistent with the capsid proteins of other T=3 plant viruses. The N-terminal arms of the A subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, have a conformation very different from the conformations observed in B and C subunits that constitute hexameric capsomers with near 6-fold symmetry at the icosahedral 3-fold axes. An analysis of the interfacial contacts between protein subunits indicates that the hexamers are held more strongly than pentamers and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. These observations suggest a plausible mechanism for the formation of empty capsids, which might be initiated by a change in the conformation of the N-terminal arm of the A subunits. The structure also provides insights into immunological and mutagenesis results. Comparison of PhMV with the sobemovirus, sesbania mosaic virus reveals striking similarities in the overall tertiary fold of the coat protein although the capsid morphologies of these two viruses are very different.

Amino Acid Sequence↗
PubMed1999

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase.

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.

Arginine↗
PubMed1999

Cavity-creating mutation at the dimer interface of Plasmodium falciparum triosephosphate isomerase: restoration of stability by disulfide cross-linking of subunits.

Disulfide engineering across subunit interfaces provides a means of inhibiting dissociation during unfolding of multimeric enzymes. Two symmetry-related intersubunit disulfide bridges were introduced across the interface of the dimeric enzyme triosephosphate isomerase from Plasmodium falciparum. This was achieved by mutating a tyrosine residue at position 74 at the subunit interface to a cysteine, thereby enabling it to form a covalent cross-link with a pre-existing cysteine at position 13 of the other subunit. The wild-type enzyme (TIMWT) and the oxidized (Y74Cox) and reduced (Y74Cred) forms of the mutant have similar enzymatic activity, absorption, and fluorescence spectra. All three proteins have similar far-UV CD spectra. The Y74Cred shows a distinct loss of near-UV CD. Thermal precipitation studies demonstrate that TIMWT and Y74Cox have very similar Tm values (Tm approximately 60 degreesC) whereas Y74Cred is surprisingly labile (Tm approximately 38 degreesC). The Y74C mutant results in the creation of a large cavity (approximately 100 A3) at the dimer interface. The crystal structure for the oxidized form of Y74C mutant, crystallized in the presence of low concentrations of dithiothreitol, reveals an asymmetric dimer containing a disulfide bridge at one site and a reduced dithiol cysteine at the other. The crystal structure of the mutant offers insights into the destabilization effects of the interfacial cavities and the role of disulfide tethering in restoring protein stability.

Animals↗
PubMed1999

On the correlation between the main-chain and side-chain atomic displacement parameters (B values) in high-resolution protein structures.

Correlation coefficients (CCs) between the mean atomic displacement parameters (B values) of the main-chain and the side-chain atoms in selected high-resolution protein structures are distributed over a broad range (0.5-1.0). The distribution of CCs is found to be related to the mean difference in B values of Calpha and Cbeta atoms. High CCs are also associated with the frequent occurrence of consecutive Calphaatoms with relatively high B values. The distribution of CCs and its relation to the mean difference between the B values of Calpha and Cbeta atoms shows dependence on the package used for refinement (X-PLOR, PROLSQ or TNT). These observations reflect the differences in the way B-value constraints are handled in these packages. Further differences are discernible in the distributions for proteins refined using the same package. It is likely that these differences are related to the different refinement protocols or weighting schemes followed by investigators. The resolution of these issues is important for evolving correct strategies for the refinement of the atomic displacement parameters in X-ray diffraction studies of proteins. Furthermore, it may be possible to develop refinement-validation tools by observing the features that are invariant in the distribution of atomic displacement parameters.

Animals↗
PubMed1999

Disulfide engineering at the dimer interface of Lactobacillus casei thymidylate synthase: crystal structure of the T155C/E188C/C244T mutant.

The crystal structure of a covalently cross-linked Lactobacillus casei thymidylate synthase has been determined at 2.8 A resolution. The sites for mutation to achieve the bis-disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.

Crystallography, X-Ray↗
PubMed1998

Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica.

Sequence analysis and metal ion binding studies reported earlier have established that the calcium binding protein (CaBP) from the parasitic ameboid Entamoeba histolytica protein has four canonical EF hand motifs which bind calcium. Equilibrium denaturation studies on both the apo and the holo forms of this protein indicate the presence of stable transition intermediates at low denaturant concentrations as revealed by the binding of the non-specific hydrophobic dye ANS. Fast reaction kinetics shows that the binding of the Gdn+ ions at or near the Ca2+ sites in the N-terminal domain influences metal ion binding to the sites in the C-terminal domain. Isothermal calorimetric titrations performed using low GdnHCl concentrations reveal the presence of two binding sites of low affinity, both being endothermic in nature. Thus the stabilization of CaBP observed at low GdnHCl concentration represents a native-like intermediate, with the Gdn+ ions mimicking Ca2+ binding at the N-terminal domain of this protein.

Animals↗
PubMed1998

Crystallization and preliminary X-ray studies of a recombinant calcium-binding protein from Entamoeba histolytica.

A calcium-binding protein (CaBP) of Entamoeba histolytica was purified from an E. coli recombinant clone carrying the CaBP gene in a pET-3c expression vector using anion-exchange and size-exclusion chromatography. Examination of the amino-acid sequence of the recombinant protein suggested that it has four independent EF-hand motifs. The protein dissolved in cacodylate buffer was crystallized using the hanging-drop method with 2-methylpentane-2,4-diol (MPD) as the precipitant. X-ray diffraction data have been collected on these crystals using a MAR Research imaging-plate detector system attached to a Rigaku RU200 rotating-anode X-ray generator. The crystals belong to the hexagonal space group P6122 with unit-cell dimensions of a = b = 96.21, c = 65.48 A. Preliminary molecular-replacement computations suggest that the structure of this protein is likely to be similar to that of calmodulin (CAM).

Amino Acid Sequence↗
PubMed1997

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica.

The thermodynamics of the binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites. There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5,5'-Br2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein. The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium. Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites. These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss of cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCl in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

Amino Acid Sequence↗
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

Detection of the peripheral nervous system (PNS)-type glial fibrillary acidic protein (GFAP) and its mRNA in human lymphocytes.

Glial fibrillary acidic protein (GFAP), an astroglial marker, has been detected in the peripheral nervous system (PNS) in a shorter version and its mRNA in a longer form (beta-type) than the brain alpha-type. To determine the characteristics of the GFAP gene expression in nonneural cells, we have investigated its in vivo transcription and translation products in human lymphocytes. Using RT-PCR, we demonstrate that the GFAP gene is transcribed in these cells. Most or all of the mRNA resulting from this transcription was longer than the brain-type at its 5' end and thus may correspond to the beta-type. In addition, immunoblotting of lymphocyte extracts with a monoclonal antibody revealed a 41 KDa fragment instead of the 50 KDa expected from brain GFAP. These results suggest that GFAP expression in lymphocytes is preferentially of the PNS beta-type giving rise to longer mRNA and shorter protein. However, compared to two other astroglial mRNAs (S-100beta and aldolase C) which were synthesized in significant amounts in lymphocytes, GFAP mRNA was detected in minute amounts representing 0.03% of the brain level. This low expression may subserve a special role in lymphocytes since it is translated.

Antibodies, Monoclonal↗
PubMed1997

Analysis of temperature factor distribution in high-resolution protein structures.

The temperature factors obtained from X-ray refinement of proteins at high resolution show large variations from one structure to another. However, the B-values expressed in units of standard deviation about their mean value (B'-factor) at the C alpha atoms show remarkably characteristic frequency distribution. In all of the 110 proteins examined in this study, the frequency distribution exhibited a bimodal distribution. The peaks in the B'-factor frequency distribution occur at -1.1 and 0.4 for a bin size of 0.5. The peak at lower temperature factor corresponds largely to buried residues, whereas the peak at larger value corresponds to exposed residues. The distribution could be accurately described as a superposition of two Gaussian functions. The parameters describing the distribution are therefore characteristic of protein structures. The frequency distribution for a given amino acid over all the proteins also shows a similar bimodal distribution, although the areas under the two Gaussians differ from one amino acid to another. The area under the frequency distribution curve for any interval in B'-factor represents the propensity of the amino acid to occur in that interval. This propensity is related both to the hydrophilicity/hydrophobicity of the residue and the tendency of the residue to impose a different degree of rigidity on the polypeptide chain. The frequency distribution of stretches of high B'-factors departs appreciably from that expected for a random distribution. The correlation in the B-values of sequentially proximal residues is probably responsible for the bimodal distribution.

Amino Acids↗