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PubMed1985

NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme.

Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1986

Structure of the copper cluster in canine hepatic metallothionein using X-ray absorption spectroscopy.

The metal binding site in the lysosomal copper metallothionein from canine liver (LyCuLP) was examined with X-ray edge and extended X-ray absorption fine structure (EXAFS) spectroscopies. The k-absorption edge spectrum of LyCuLP was consistent with the coordination of univalent copper. The Fourier transform of the EXAFS data showed four resolved shells of backscattering atoms. Comparisons between the phase and amplitude functions derived from the isolated shells to those of Cu-Cu, Cu-S, and Cu-N model compounds showed that each copper was coordinated by four sulfur atoms at a distance of 2.27 +/- 0.02 A. Analysis of the outer shell data indicated backscattering copper atoms at 2.74 +/- 0.05, 3.32 +/- 0.05, and 3.88 +/- 0.05 A. Interatomic distances determined from the EXAFS data were compared to the distances observed by X-ray crystallographic analysis of adamantane-like clusters containing four and five copper atoms and a cubic cluster containing four copper atoms, structurally similar to the 4Fe-4S clusters in some ferredoxins. The results of these comparisons suggest that the copper complexed in LyCuLP is arranged in an adamantane-like cluster. The structure derived for this protein may be conserved in other copper metallothioneins.

Animals↗

PubMed1989

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.

Base Sequence↗

PubMed1990

2D NMR studies and 3D structure of the parallel-stranded duplex oligonucleotide Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The three-dimensional structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAACTC) covalently linked to an acridine derivative (m5Acr) with its natural and parallel complementary sequence beta-d(AGATTTGAG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. All the nonexchangeable and exchangeable resonances were assigned in this duplex. The structure was refined by using interproton distances determined by NOE measurements. The NOE values were converted into distances by using the complete 190 x 190 relaxation matrix. The unnatural duplex Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) forms a parallel right-handed helix with Watson-Crick base pairing; the alpha and beta deoxyriboses adopt a 3'-exo conformation. The acridine moiety was found stacked up the C9-G9 base pair. The structure of the first seven base pairs of this duplex was found similar to that of the duplex alpha-d(TCTAAAC)-beta-d(AGATTTG), which we had already investigated [Lancelot, G., et al. (1989) Biochemistry 28, 7871-7878]. Since these structures were generated by using experimental NOE values obtained independently on macromolecules whose global correlation time was different (3.8 and 2.2 ns), we conclude that this comparison is a good test of the viability of our method to generate three-dimensional structures of oligonucleotides in solution. Starting from different initial conformations, we show that the NOE constraints allow one to reach the same final restrained conformation, taking into account implicitly the solvent effect.

Aminoacridines↗

PubMed1975

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.

Animals↗

PubMed1991

Protein solution structure determination using distances from two-dimensional nuclear Overhauser effect experiments: effect of approximations on the accuracy of derived structures.

Solution structures for many proteins have been determined to date utilizing interproton distance constraints estimated from two-dimensional nuclear Overhauser effect (2D NOE) spectra. Although the simple isolated spin pair approximation (ISPA) generally used can result in systematic errors in distances, the large number of constraints enables protein structure to be defined with reasonably high resolution. Effects of these systematic errors on the resulting protein structure are examined. Iterative relaxation matrix calculations, which account for dipolar interactions between all protons in a molecule, can accurately determine internuclear distances with little or no a priori knowledge of the molecular structure. The value of this additional complexity is also addressed. To assess these distance determination methods, hypothetical "experimental" data, including random noise and peak overlap, are calculated for an arbitrary "true" protein structure. Three methods of obtaining distance constraints from 2D NOE peak intensities are examined: one entails a conservative use of ISPA, one assumes the ISPA to be fairly accurate, and one utilizes an iterative relaxation matrix method called MARDIGRAS (matrix analysis of relaxation for discerning the geometry of an aqueous structure), developed in this laboratory. A distance geometry algorithm was used to generate a family of structures for each distance set. The quality of the average structure from each family was good. The root-mean-square deviation of that average structure from the true structure was improved about 2-5% using the more restrictive rather than the more conservative ISPA approach. Use of MARDIGRAS in a conservative fashion--i.e., with a poor initial model--resulted in improvement in the root-mean-square deviation by 8-15%. With a better initial model, MARDIGRAS obtained even more accurate distances. MARDIGRAS also permits analysis of 2D NOE data at longer mixing times, yielding additional distances. Use of more restrictive ISPA distances did, however, result in a few systematically incorrect structural features in local regions of the protein, producing distortions of 2-3 A. Comparison between experimental data and spectra calculated for the structures correlates with root-mean-square deviation, offering a method of structure evaluation. An R factor for evaluating fit between experimental and calculated 2D NOE intensities is proposed.

Magnetic Resonance Spectroscopy↗

PubMedDate not supplied

The geographic clustering of body size of Chinese children aged 7 years.

We studied the morphological differences and similarities among Chinese Han children aged 7 years by using PCA-Q mode cluster analysis. The data were drawn from 17,119 boys and 17,084 girls who came from 28 provinces and participated in the 1985 National Survey on Students Constitution and Health. The children were divided into urban and rural subgroups in each province. First, the values of six measures of body size (stature, sitting height, body weight, chest girth, biacromial diameter and biiliocristal diameter) were normalized into principal component scores; and then clustering was performed among subgroups according to the distances determined by the component scores. The results show significant variations in children's body size exist not only between north and south China, but also between developed and under-developed districts and between urban and rural areas. The geographic and socio-economic factors causing these variations were investigated. The practical use of PCA-Q cluster analysis and its merits in studying Chinese children's growth variations are discussed.

Anthropometry↗

PubMed1987

An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations.

We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.

Algorithms↗

PubMed1988

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila.

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.

Alcohol Dehydrogenase↗

PubMed1990

Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A 1H- and 31P-NMR investigation.

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spin-lattice relaxation rates (T-1(1] of ligand nuclei (1H and 31P) in E.Mn(II).Glc and E.Mn(II).Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H alpha, C1-H beta and C2-H beta of glucose in the E.Mn(II).Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30 degrees C at 500 MHz. The ligand nuclear relaxation rates in E.Mn(II).Glc are field-dependent and the Arrhenius plot yields an activation energy (delta E) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H alpha, C1-H beta and C6-31P at 270 MHz (1H) and 202.5 MHz (31P) for the E.Mn(II).Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5-30 degrees C, which yielded delta E = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (tau c), determined from the field-dependent measurements of proton relaxation rates in the E.Mn(II).Glc complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are approximately 1.1 nm and approximately 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P.Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of approximately 0.5-0.6 nm from the cation.

Animals↗

PubMed1988

Molecular phylogeny of the animal kingdom.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Animals↗

PubMed1966

Fine-structure mapping by transformation in the tryptophan region of Bacillus subtilis.

Carlton, Bruce C. (Yale University, New Haven, Conn.). Fine-structure mapping by transformation in the tryptophan region of Bacillus subtilis. J. Bacteriol. 91:1795-1803. 1966.-The arrangements of eight mutational sites within a segment of the tryptophan cluster of Bacillus subtilis have been determined by three-point reciprocal crosses with a mutation in the same gene cluster used as an outside marker. Estimates of distances between sites were made by two-point crosses and normalization to recombination values for an unlinked marker to correct for physiological variabilities between recipient strains. In general, the additivities of map distances determined by this procedure were reasonable, although some discrepancies were noted in reciprocal crosses between widely separated sites within the gene region examined. No definitive polarity effect on recombination was evident, and it appears that the variations observed in reciprocal crosses may be an effect of the probability of recovery of crossovers between exogenotic and endogenotic segments. It is concluded that fine-structure mapping is feasible for the transformation system in this organism and that a high degree of resolution can be attained.

Bacillus subtilis↗

PubMed1992

Cuticular hydrocarbons, isoenzymes and behavior of three populations of Anopheles darlingi from Brazil.

Three populations of Anopheles darlingi were studied for cuticular hydrocarbons, isoenzymes and patterns of peak biting activity. Differences were found in specimens from Costa Marques, a malaria endemic area; Dourado, a site with a very exophilic population and Juturnaíba, located near the type locality. Twelve hour collections from sunset to sunrise showed that An. darlingi from Costa Marques had a bimodal biting activity profile with a major peak at sunset and a minor peak at sunrise. At Dourado, the pattern was trimodal, with peaks at both morning and evening periods of twilight and near midnight. The Juturnaíba population showed a slight increase in activity near 2000 and 0100 h. Nei's genetic distances, determined by isoenzyme electrophoresis between pairs of populations, were low (D < or = 0.049). Using discriminant analysis for the cuticular hydrocarbons, 92.4% of the specimens from Costa Marques, 91.2% of the specimens from Dourado and 61.3% from Juturnaíba were correctly identified. Cuticular hydrocarbon and isoenzyme results matched very well: the smaller the Nei's distance, the more misidentifications occurred in the jackknife estimator used in the cuticular hydrocarbon analysis. This is the first report of cuticular hydrocarbon analysis in combination with isoenzymes to investigate neotropical anopheline species.

Animals↗

PubMed1991

Arrangement of the substrates at the active site of brain pyridoxal kinase.

The distances between enzyme-bound paramagnetic CrATP (a stable, beta, gamma-bidentate complex of Cr3+ and ATP) at the active site of sheep brain pyridoxal kinase and the protons of bound inhibitor 4-dPyr (4-deoxypyridoxine) were determined in the ternary enzyme-CrATP.4-dPyr complex by measuring the paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the protons of 4-dPyr. The correlation time for the Cr(3+)-4-dPyr dipolar interaction on the enzyme was estimated as 1.59 ns by the frequency dependence of 1/T1p of water protons. Temperature dependence of 1/T1p values indicated the fast exchange of 4-dPyr from the paramagnetic enzyme.CrATP.4-dPyr complex; hence the measured 1/T1p values can be used for metalnucleus distance determinations. The distances from the Cr3+ of the enzyme-bound CrATP to the 2-methyl (7.19 A), 4-methyl (7.18 A), and H6 proton (6.18 A) of the 4-dPyr are too great to permit a direct coordination of any group from 4-dPyr. However, these distances can be built into a model in which phosphorus of the gamma-phosphoryl group of ATP is 4 A away from the oxygen atom of the 5-CH2OH group of the 4-dPyr. This suggests that phosphorylation of pyridoxal can occur via direct transfer of the phosphoryl group between the bound substrates at the active site of pyridoxal kinase.

Adenosine Triphosphate↗

PubMed1990

Identification of a beta-turn in the tertiary structure of a peptide fragment of the Alzheimer amyloid protein.

In an attempt to understand the three-dimensional structure of the Alzheimer amyloid protein, a fragment decapeptide was studied by nuclear magnetic resonance spectroscopy. Using two-dimensional techniques, protons close in space were identified and their interproton distances determined. These distances were then used as input data for distance geometry calculations. The resulting three-dimensional structures suggest the presence of a beta-turn in the C-terminus of the decapeptide.

Amino Acid Sequence↗

PubMed1989

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification.

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.

Allosteric Regulation↗

PubMed1985

Nuclear magnetic resonance studies of fructose 2,6-bisphosphate and adenosine 5'-monophosphate interaction with bovine liver fructose-1,6-biphosphatase.

1H and 31P nuclear magnetic resonance was used to investigate the interaction of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) with bovine liver fructose-1,6-bisphosphatase. Mn2+ bound to fructose-1,6-bisphosphatase was used as a paramagnetic probe to map the active and AMP allosteric sites of fructose-1,6-bisphosphatase. Distances between enzyme-bound Mn2+ and the phosphorus atoms at C-6 of fructose-6-P and alpha-methyl-D-fructofuranoside 1,6-bisphosphate were identical, and the enzyme-Mn to phosphorus distance determined for the C-6 phosphorus atom of Fru-2,6-P2 was very similar to these values. Likewise, the enzyme-Mn to phosphorus distances for Pi, the C-1 phosphorus atom of alpha-methyl-D-fructofuranoside 1,6-bisphosphate, and the C-2 phosphorus atom of Fru-2,6-P2 agreed within 0.5 A. The distance between enzyme-bound Mn2+ and the phosphorus atom of AMP was significantly shorter than the distances obtained for any of the aforementioned ligands, but the presence of Fru-2,6-P2 caused the enzyme-Mn to phosphorus distance for AMP to lengthen markedly. NMR line broadening of AMP protons was studied at various temperatures. The dissociation rate constant was found to be greater than 20 s-1. It was concluded that Fru-2,6-P2 strongly affects the interaction of AMP with fructose-1,6-bisphosphatase and that the sugar most likely acts at the active site of the enzyme.

Adenosine Monophosphate↗

PubMed1986

[Analysis of the reproducibility of conditions of intracavitary irradiation of patients with cervix cancer].

The paper is concerned with the results of an analysis of reproducibility of conditions for intracavitary irradiation of 20 cervical cancer patients using metracolpostat, a combined therapeutic endostat consisting of 3 tubes: the central one to introduce radiation sources into the cervical canal and cavity of the uterus and 2 lateral ones with ovoids to deliver the sources to the vaults of the vagina. Topometric investigations for each patient were performed 4 times: before the first session of fractionated intracavitary irradiation with high activity 252Cf sources using the ANET-B unit and after each session of irradiation. It was shown that discordances between distances from the central endostat to the right and left lateral ovoids determined during roentgenotopometric preparation of each patient and taken as a basis of dosimetric design, and discordances between these distances determined as a result of roentgenotopometric control after each laying, are a random value distributed according to the normal law.

Brachytherapy↗