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B D Rao

Publications and source records attributed to B D Rao.

At least 19 recordsLinked to original sources
PubMed1997

Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain.

Adenosine↗
PubMed1996

Conformation of manganese(II)-nucleotide complexes bound to rabbit muscle creatine kinase: 13C NMR measurements using [2-13C]ATP and [2-13C]ADP.

Conformations of cation-nucleotide complexes bound to rabbit muscle creatine kinase were investigated by measuring paramagnetic effects on 13C spin relaxation in E.Mn[2-13C]ATP and E.Mn[2-13C]ADP at three different frequencies, viz., 50, 75, and 125 MHz, and as a function of temperature in the range of 7-35 degrees C (at 75 MHz). Arrhenius plots of the temperature dependencies of relaxation rates show a positive slope with low activation energies of 1.3 +/- 0.2 kcal/mol and 2.0 +/- 0.2 kcal/mol for E.Mn ATP and E.MnADP, respectively. The relaxation rates of both complexes show strong frequency dependence, indicating that these rates are not exchange limited. Analysis of the data yields Mn(II)-2C distances of 10.0 +/- 0.5 A for E.MnATP and 8.6 +/- 0.5 A for E.MnADP. These data were interpreted, along with previously published information, on the location of the cation with respect to the phosphate chain [Jarori, G. K., Ray, B.D., & Nageswara Rao, B. D. (1985) Biochemistry 24, 3487-3494], and on the adenosine conformation [Murali, N., Jarori, G. K., & Nageswara Rao, B. D. (1993) Biochemistry 32, 12941-12948] in these complexes. The Mn(II)-2C distances depend on the orientation of the phosphate chain relative to the adenosine moiety. Conformational searches were performed by varying the two torsion angles, phi 1 (C4'-C5'-O5'-P alpha), and phi 2 (C5'-O5'-P alpha-O alpha beta), along with CHARMm energy computations, in order to determine acceptable conformations compatible with the distances determined. The significant difference in the Mn(II)-2C distances in E.MnATP and E.MnADP is indicative of the structural alterations occurring at the active site as the enzyme turns over.

Adenosine Diphosphate↗
PubMed1995

Conformation of MgATP bound to 5-phospho-alpha-D-ribose 1-diphosphate synthetase by two-dimensional transferred nuclear Overhauser effect spectroscopy.

The conformation of MgATP bound at the active site of Salmonella typhimurium 5-phospho-alpha-D-ribose 1-diphosphate synthetase (PRibPP synthetase) has been investigated by two-dimensional transferred-NOE spectroscopy (TRNOESY). Inter-proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40-300 ms at 500 MHz and 10 degrees C. Measurements were made at low ligand concentrations (approximately 1 mM) in order to avoid weak non-specific ligand-protein interactions and to ensure that the NOE arises from the ligand bound at the active site. The inter-proton distances were determined from the experimentally observed NOE buildup curves by comparing them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand. PRibPP synthetase is known to appreciably aggregate so that it exists in multiple oligomeric forms in solution. The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size. On the basis of the rotational correlation time deduced for the enzyme-nucleotide complexes, it is estimated that the average oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer. The conformation of enzyme-bound MgATP is described by a glycosidic torsion angle chi = 50 +/- 5 degrees and phase angle of pseudorotation P = 114.9 degrees corresponding to a 1T degree sugar pucker. It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase. The sugar pucker, on the other hand, differs from one enzyme complex to another.

Adenosine Triphosphate↗
PubMed1995

Neuromagnetic source imaging with FOCUSS: a recursive weighted minimum norm algorithm.

The paper describes a new algorithm for tomographic source reconstruction in neural electromagnetic inverse problems. Termed FOCUSS (FOCal Underdetermined System Solution), this algorithm combines the desired features of the two major approaches to electromagnetic inverse procedures. Like multiple current dipole modeling methods, FOCUSS produces high resolution solutions appropriate for the highly localized sources often encountered in electromagnetic imaging. Like linear estimation methods, FOCUSS allows current sources to assume arbitrary shapes and it preserves the generality and ease of application characteristic of this group of methods. It stands apart from standard signal processing techniques because, as an initialization-dependent algorithm, it accommodates the non-unique set of feasible solutions that arise from the neuroelectric source constraints. FOCUSS is based on recursive, weighted norm minimization. The consequence of the repeated weighting procedure is, in effect, to concentrate the solution in the minimal active regions that are essential for accurately reproducing the measurements. The FOCUSS algorithm is introduced and its properties are illustrated in the context of a number of simulations, first using exact measurements in 2- and 3-D problems, and then in the presence of noise and modeling errors. The results suggest that FOCUSS is a powerful algorithm with considerable utility for tomographic current estimation.

Algorithms↗
PubMed1994

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in arginine kinase complexes.

Two-dimensional proton transfer nuclear Overhauser spectroscopy (TRNOESY) studies of the conformations of the adenosine moieties in the nucleotides bound at the active site of the lobster (Homarus americanus) muscle arginine kinase are reported. TRNOESY measurements were made using a sample protocol chosen to minimize contributions from weak non-specific binding of the nucleotides to the observed NOE's. This was done by making the measurements as a function of ligand concentration while keeping the ligand to enzyme concentration ratio fixed at 10:1. The experiments were performed at 500 MHz and 10 degrees C for six different mixing times in the range 40-300 ms. The measurements were made on three complexes of the enzyme: E.MgATP, E.MgADP, and the long-lived transition-state-analog complex (E.MgADP.NO-3.arginine). All the complexes, including the transition-state-analog complex with an estimated lifetime of about 50 ms, satisfy the fast-exchange condition. The TRNOE buildup curves for all the nucleotide-proton pairs in each complex were analyzed using a complete relaxation matrix appropriate for fast exchange. The interproton distances obtained from the NOE analysis were used as constraints in obtaining an energy-minimized conformation on the basis of the program CHARMm. The glycosidic torsion angle (chi) for the adenosine moiety in all three complexes is about 50 degrees +/- 5 degrees. The glycosidic orientation agrees well with that determined for MgATP and MgADP complexes of creatine kinase (Murali et al., 1993), MgATP bound at the active and ancillary sites of pyruvate kinase (Jarori et al., 1994a), and PRPP synthetase (Jarori et al., 1994b).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1994

Two-dimensional transferred nuclear Overhauser effect spectroscopy study of the confirmation of MgATP bound at the active and ancillary sites of rabbit muscle pyruvate kinase.

Pyruvate kinase binds one adenosine 5'-triphosphate (ATP) molecule at its active site and another at an ancillary site on each subunit. In order to determine the conformation of ATP bound at these sites, proton transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements were made at 500 MHz and 10 degrees C for several mixing times in the range 40-200 ms. The NOE values for the proton pair H1'-H2' of ribose (which are 2.9 +/- 0.2 A apart, irrespective of nucleotide conformation) as a function of ligand concentration (1-10 mM ATP), with the ratio of ligand to enzyme being kept constant, indicate that at higher ligand concentrations adventitious binding of ATP at nonspecific site(s) makes a major contribution to the observed NOEs. When the ligand concentration is < 2 mM, site-specific NOEs can be measured. Furthermore, addition of phosphoenolpyruvate (PEP) to the enzyme-MgATP sample results in competitive displacement of MgATP from the active site and reduces the observed NOE to that arising exclusively at the ancillary site, thus allowing the measurement of site-specific NOEs. The interproton distances determined from such site-specific NOE buildup curves were used as constraints in CHARMm to obtain the structure of MgATP. At the active site, MgATP has a glycosidic torsion chi = 44 +/- 5 degrees and the phase angle of pseudorotation for ribose P = 42.4 degrees. At the ancillary site chi = 46 +/- 5 degrees and P = 127.6 degrees. Thus the orientation of the adenine with respect to the sugar moiety is the same at both sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) studies of nucleotide conformations in creatine kinase complexes: effects due to weak nonspecific binding.

The conformations of the adenosine moiety of MgADP and MgATP bound to rabbit muscle creatine kinase were investigated by two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The effects arising from adventitious binding of the ligands to the enzyme on the measurements were delineated. It was shown that, with sample protocols typically used thus far with the TRNOE method (enzyme, approximately 1 mM; ligand, approximately 10 mM), the TRNOESY pattern for the nucleotides with creatine kinase is similar to that with gamma-globulin and bovine serum albumin, which do not have specific nucleotide binding site(s). Measurements of NOE between the H1'-H2' proton pair as a function of ligand concentrations with the enzyme-ligand ratio kept constant at 1:10 showed that, for ligand concentrations over about 3-4 mM, weak nonspecific binding makes a significant contribution to the observed NOE. Thus the NOE values relevant for the determination of the nucleotide conformation at the active site were measured at nucleotide concentrations of about 1.5 mM. The TRNOE buildup curves for all the ligand-proton pairs were analyzed using a complete relaxation matrix approach. The interproton distances derived from the NOE's were then used as constraints in elucidating the ligand structure by using the program CHARMm. The NOE-determined structures of both MgADP and MgATP bound to creatine kinase correspond to an anti conformation with the glycosidic angle (O'4-C'1-N9-C8) chi = 51 +/- 5 degrees. The ribose pucker nominally representative of these data is a O4'T with a phase angle of pseudorotation (p) of 70.5 degrees.

Adenosine↗
PubMed1992

Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes 1H-transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase.

The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 1H NMR. The experiments were performed on D2O solutions at 276 MHz and 300 MHz, and 10 degrees C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase-alternating hard 1.8 degree pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6-8 delay times ranging from 10-500 ms following the DANTE sequence. A full complement of ten NOE build-up curves obtained for each enzyme complex was analyzed by using the complete relaxation-matrix method (which includes all the non-exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE-determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3'-endo sugar pucker and an anti conformation with a glycosidic torsional angle (O'4-C'1-N9-C8) of 39 degrees +/- 4 degrees. Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth-power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P.R., Fox, T.L. & Mildvan, A.S. (1987) Biochemistry 26, 3487-3493.

Adenine↗
PubMed1991

Determination of in vitro simulated blood cell velocity by laser-Doppler with state space methods.

In this paper a signal processing method based on an important tool of numerical analysis, the singular value decomposition (SVD), is used. This method is applied to the unprocessed output of a Laser-Doppler fluxmeter to obtain parameters that are more sensitive to blood velocity than to hematocrit. The SVD-based method utilizes the exponential shape of the frequency spectrum of the laser light scattered from the moving red blood cells and demonstrates an inverse relationship between the damping constant beta associated with the exponential shape and the blood velocity. This method was applied to samples of rat blood of several known hematocrit values that were rotated at different velocities on a turntable. The method extracted one dominant singular value from the spectra, indicating that the spectra can be modeled as a single exponential dominated by scatterings with a single moving red blood cell. A 68% change in inverse velocity resulted in a 50% change in the damping constant (hematocrit 29.5% vol). On the other hand, a 37% change in hematocrit resulted in a far smaller change in the damping constant beta of only 17%. Analysis of the extracted parameters shows that the damping constants are far more influenced by blood velocity than by blood hematocrit.

Animals↗
PubMed1990

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase: III. Two ADP binding sites and their Mg(II) affinity; effects of vanadate and arsenate on enzymic complexes with ADP and 3-P-glycerate.

31P nuclear magnetic resonance (NMR) measurements (at 121.5 MHz and 5 degrees C) were made on complexes of 3-phosphoglycerate kinase with ADP and 3-P-glycerate. Addition of Mg(II) to E.ADP shifts the alpha-P signal downfield by 3.8 ppm such that the alpha-P signal superimposes that for beta-P(E.MgADP). Such a shift is atypical among the Mg(II)-nucleotide complexes with other ATP-utilizing enzymes. This shift allowed the determination that enzyme bound ADP is saturated with Mg(II) for [Mg(II)]/[ADP] = 3.0--similar to that reported for ATP complexes with this enzyme (B.D. Ray and B.D. Nageswara Rao, Biochemistry 27, 5574 (1988]. This parallel behavior suggests that ADP binds at two sites on the enzyme as does ATP with disparate Mg(II) affinities. 31P relaxation times in E.MnADP.vanadate.3-P-glycerate and E.CoADP.vanadate.3-P-glycerate complexes indicate that these are long-lived, tightly bound complexes. 31P chemical shift measurements on diamagnetic complexes (with Mg(II] revealed three signals in the 2-5 ppm region (attributable to 3-P-glycerate) only upon addition of all the components necessary to form the E.MgADP.vanadate.3-P-glycerate complex. Subsequent sequestration of Mg(II) from the complex with excess EDTA reversed the Mg(II) induced effects on the ADP signals but did not cause coalescence of the three signals seen in the 2-5 ppm region. Addition of excess sulfate to dissociate these complexes from the enzyme resulted in a single resonance of 3-P-glycerate. The use of arsenate in place of vanadate yielded very similar results. These results suggest that, in the presence of MgADP, vanadate or arsenate, and 3-P-glycerate, the enzyme catalyzed the formation of multiple structurally distinguishable complexes that are stable on the enzyme and labile off the enzyme.

Adenosine Diphosphate↗
PubMed1989

Nuclear magnetic resonance line-shape analysis and determination of exchange rates.

The fact that chemical exchange processes occur at rates that cover a broad range and produce readily detectable effects on the spectrum is one of the attractive features of high-resolution NMR. The description of these line shapes in the presence of spin-spin coupling requires the density matrix theory which is rather complex. Analysis of the line shapes usually needs computer simulations and is capable of providing reliable information on the exchange rates as well as spectral parameters in the absence of exchange. Simplified procedures, ignoring spin-spin coupling, often result in deviations in these exchange and spectral parameters determined. A step-by-step procedure is detailed in this chapter for setting up the matrices required for computing the line shapes of exchanges involving weakly coupled spin systems on the basis of the density matrix theory without the need for a detailed understanding of the theory. A knowledge of the energy level structure and allowed transitions in the NMR spectra of the individual weakly coupled spin systems is all that is required. The procedure is amenable to numerical computation. The group of illustrative examples chosen to demonstrate the development of the computational tools cover some of the commonly encountered cases of exchange from simple systems to rather complex ones. Such exchanges occur frequently in biological molecules, especially those involving enzyme-substrate complexes. In cases where the experimental line shapes are obtained with respectable precision, and the relevant exchange processes are unambiguously identifiable, the computer simulation method of line-shape analysis is capable of providing useful and incisive information. The example of the 31P exchanges in the adenylate kinase is illustrative of this point. Not only has the line-shape analysis clearly indicated the role of the interchange process, but it has also produced evidence that the rates of interchange of the ADP molecules bound to the enzyme become relevant to the kinetics and mechanism of catalysis by this enzyme. It is probably difficult to obtain this information by any other experimental method or by any other method of analysis.

Adenylate Kinase↗
PubMed1988

31P NMR studies of the structure of cation-nucleotide complexes bound to porcine muscle adenylate kinase.

The paramagnetic effects on the spin-relaxation rates of 31P nuclei in complexes of porcine muscle adenylate kinase with ATP, GTP, GDP, and AMP were measured in the presence of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), to examine the structures of the enzyme-bound complexes. Experiments were performed exclusively on enzyme-bound complexes to limit contributions to observed relaxation rates to two exchanging complexes (with and without cation). Measurements were made at three frequencies, 81, 121.5, and 190.2 MHz, and as a function of temperature in the range 5-30 degrees C to determine the effect of exchange on the observed relaxation rates. Relaxation rates in the E.MnATP, E.MnGTP, and E.MnGDP complexes were shown to be exchange-limited and therefore without structural information. Relaxation rates for the complexes E.CoATP, E.CoGTP, and E.CoGDP were shown to depend on Co(II)-31P distances. Inability to precisely estimate spectral densities arising from electronic relaxation of Co(II) restricts calculations of Co(II)-31P distances in these complexes to upper and lower limits. At the center of these limits, the Co(II)-31P distances of beta-P and gamma-P in E.CoATP and E.CoGTP, and of beta-P (E.CoGDP), are in the range 3.1-3.5 A appropriate for the first coordination sphere. For all these complexes, the corresponding distance for alpha-P is appreciably larger in the range 3.9-4.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1988

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 1. Effects of sulfate and pH. Mg(II) affinity at the two ATP sites.

31P NMR measurements were made (at 121.5 MHz and 5 degrees C) on enzyme-bound substrate complexes of 3-phosphoglycerate kinase in order to address three questions pertaining to (i) the integrity of the enzyme-substrate complexes with Mg(II) in the presence of sulfate concentrations typical of those used for crystallization in X-ray studies, (ii) the relative affinities of Mg(II) to ATP bound at the two sites on the enzyme, and (iii) the pH behavior of the different phosphate groups in the enzyme complexes. 31P chemical shift and spin-spin coupling constant changes showed that at concentrations of 0.5 M and higher, sulfate ion interferes with Mg(II) chelation to ATP and ADP free in solution as well as in their enzyme-bound complexes. The effect on enzyme complexes is stronger for the E.MgATP complex than for the E.MgADP complex. Sulfate ion (50 mM) also causes a approximately 0.5 ppm upfield chemical shift of the 31P resonance of enzyme-bound 3-P-glycerate even in the absence of ATP or Mg(II). A quantitative estimate of the dispartate affinities of Mg(II) to ATP bound at the two sites on the enzyme was made on the basis of computer simulation of changes in the line shape of beta-P (ATP) resonance and of changes in 31P chemical shift of the corresponding gamma-P (ATP) in the E.ATP complex with increasing [Mg(II)]. The concentrations of the relevant species that contribute to these 31P NMR signals were computed by assuming independent binding at the two sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 2. Structure measurements using paramagnetic relaxation effects of Mn(II) and Co(II).

Measurements of the paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin relaxation rates of the 31P nuclei in the complexes of 3-phosphoglycerate kinase with ATP, ADP, and 3-P-glycerate have been used to study the structures of these enzyme-substrate complexes. All experiments were performed on enzyme-bound complexes, so that two exchanging complexes (with and without cation) contribute to the observed relaxation rate. Measurements were made at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, and as a function of temperature in the range 5-20 degrees C to determine the effect of exchange on the observed relaxation rates. Relaxation rates in E.MnADP and E.MnATP were shown to be exchange-limited, and therefore bereft of structural information, both by lack of frequency dependence and by temperature dependence with activation energies (delta E) in the range 5-8 kcal/mol. Relaxation rates for E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances. Difficulties involved in estimating electron relaxation times in E.CoADP and E.CoATP restrict calculation of Co(II)-31P distances in these complexes to upper and lower limits. These distances were all in the range 2.7-4.1 A, appropriate for direct coordination of Co(II) to the phosphate groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1984

Electron paramagnetic resonance of spin-labeled aequorin.

Aequorin is a Ca-activated bioluminescent protein from jellyfish. This protein contains two sulfhydryl groups, one of which is essential for its bioluminescence. Little information concerning the structure of and relationship between the metal binding sites of aequorin and the sulfhydryl group(s) is known. Aequorin was modified by attachment of either a maleimide spin-label [studied by electron paramagnetic resonance (EPR)] or the fluorescent label Acrylodan at the essential sulfhydryl in order to gain such information. These modifications caused destabilization of the chromophore of aequorin. Both of the attached labels showed considerable freedom of motion. The spin-label was quite accessible to the solvent, and the fluorescent label was less so. In addition the metal binding properties of the spin-labeled aequorin were studied by Mn(II) EPR. One tight Mn(II) binding site per spin-labeled aequorin was found. The distance between the Mn(II) binding site and the spin-label is at least 20 A. Furthermore, the relative affinity of spin-labeled aequorin for various metal ions was found to be in the order Pr(III) greater than Mn(II) greater than Ca(II) greater than Mg(II).

2-Naphthylamine↗