Biomedical subjects

G M Clore

Publications and source records attributed to G M Clore.

At least 253 records · Page 14Linked to original sources

PubMed1985

The structure of the double-stranded RNA pentamer 5'(CACAG) . 5'(CUGUG) determined by nuclear Overhauser enhancement measurements: interproton distance determination and structure refinement on the basis of X-ray coordinates.

The structure in solution of the duplex RNA pentamer 5'(CACAG) . 5'(CUGUG), comprising the stem of the T psi C loop of yeast tRNAPhe, has been investigated by means of one- and two-dimensional nuclear Overhauser enhancement measurements. All non-exchangeable base and sugar proton resonances with the exception of the H5'/H5" sugar resonances are assigned in a sequential manner. From the relative intensities of the cross-peaks obtained in the pure-phase absorption two-dimensional nuclear Overhauser enhancement spectra at several mixing times, it is deduced that the RNA pentamer adopts an A-type conformation in solution. Cross-relaxation rates and interproton distances are determined from the time dependence of the nuclear Overhauser effects, principally by one-dimensional measurements. The structure of the RNA pentamer is then refined by restrained least-squares minimization on the basis of both distance and planarity restraints using fibre diffraction data as an initial model. The refined structure of the RNA pentamer is of the A type but exhibits local structural variations in glycosidic bond and backbone torsion angles as well as in propeller twist, base roll and base tilt angles.

Base Sequence↗

PubMed1985

The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices.

Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 M-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146) beta. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94) beta, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 M-NaCl + 0.2 M-phosphate this change of ligation lowered the pKa of the histidine from 8.0 in Hb to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pKa in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect. We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)beta----Leu), HbCO Wood (His FG4(97)beta----Leu) and HbCO Malmø (His FG4(97)beta----Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)beta in fact belongs to His FG4(97)beta. Although in Hb the pKa of His HC3(146)beta is 8.05 +/- 0.05 independent of ionic strength, in HbCO its pKa drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pKa of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973). In HbCO A, His FG4(97)beta does not contribute to the Bohr effect, but in HbCO from which His HC3(146)beta has been cleaved (HbCO des-His), His FG4(97)beta is in equilibrium between two conformations with different pKa values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety. In HbCO A, His FG4(97)beta has a pKa of 7.8 compared to the pKa value of about 6.6 characteristic of free histidines at the surface of proteins. This high pKa is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of alpha-helices, which manifests itself even when they lie on the surface of the protein.

Carboxyhemoglobin↗

PubMed1985

Probing the three-dimensional structures of DNA and RNA oligonucleotides in solution by nuclear Overhauser enhancement measurements.

DNA and RNA oligonucleotides are ideally suited for high-resolution X-ray crystallographic and 1H-NMR studies. The solution structures of such oligonucleotides can potentially be solved using proton-proton nuclear Overhauser enhancement measurements to demonstrate the proximity of protons in space and to determine their separation, thereby enabling a comparison of the structure in the crystalline and solution states to be made. In this review we describe the general strategy for the sequential resonance assignments of oligonucleotide 1H-NMR spectra, the essential prerequisite for further structural work, the approach to obtaining interproton distances from pre-steady state nuclear Overhauser enhancement measurements, and the use of interproton distances in structure determination. This is illustrated by several examples including double- and single-stranded DNA oligonucleotides as well as RNA stem and loop structures.

Base Sequence↗

PubMed1985

The solution structure of a B-DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon. Refinement on the basis of interproton distance data.

A restrained least squares refinement of the solution structure of the double-stranded DNA undecamer 5'd(AAGTGT-GACAT).5'd(ATGTCACACTT) comprising a portion of the specific target site of the cAMP receptor protein in the gal operon is presented. The structure is refined on the basis of both distance and planarity restraints, 2331 in all. The distance restraints comprise 150 interproton distances determined from pre-steady state nuclear Overhauser enhancement measurements and 2159 other interatomic distances derived from idealized geometry (i.e., distances between covalently bonded atoms, between atoms defining fixed bond angles, and between atoms defining hydrogen bonding in AT and GC base pairs). Two refinements were carried out and in both cases the final RMS difference between the experimental and calculated interproton distances was 0.2 A. The difference between the two refined structures is small (overall RMS difference of 0.23 A) and represents the error in the refined coordinates. Although the refined structures have an overall B-type conformation there are large variations in many of the local conformational parameters including backbone and glycosidic bond torsion angles, helical twist and propellor twist, base roll and base tilt angles.

Base Sequence↗

PubMed1985

Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei.

The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.

Base Sequence↗

PubMed1984

A two-dimensional NMR study of the solution structure of a DNA dodecamer comprising the concensus sequence for the specific DNA-binding sites of the glucocorticoid receptor protein.

A two-dimensional 500-MHz 1H-NMR study on the non-self-complementary double-stranded DNA dodecamer 5'd(C-C-A-G-A-A-C-A-G-T-G-G)5'd(C-C-A-C-T-G-T-T-C-T-G-G), is presented. This oligonucleotide contains the consensus octanucleotide sequence 5'd(A-G-A-A-C-A-G-T) for the specific DNA-binding sites of the glucocorticoid receptor protein [Payvar, F. et al. (1984) Cell 35, 381-392]. Using a combination of two-dimensional pure phase absorption nuclear Overhauser enhancement (NOESY) and homonuclear J-correlated (COSY) spectroscopy all non-exchangeable base (with the exception fo the adenine H2 protons), methyl and deoxyribose H1', H2', H2", H3' and H4' resonances are assigned unambiguously using a sequential resonance assignment strategy. From the relative intensities of the cross peaks in the pure phase absorption NOESY spectra at two mixing times it is shown that the dodecamer adopts a B-type conformation in solution.

Base Sequence↗

PubMed1984

Yeast tRNAAsp: codon and wobble codon-anticodon interactions. A transferred nuclear Overhauser enhancement study.

The conformations of the ribotrinucleoside bisphosphates GpApC and GpApU, the codon and wobble codon for aspartic acid respectively, bound to yeast tRNAAsp in solution, have been examined by means of time-dependent transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The conformations of the two bound ribotrinucleoside bisphosphates are shown to be very similar with an overall root-mean-square difference in interproton distances of 0.03 nm. The ribose conformations of all the residues are 3'-endo; the glycosidic bond torsion angles of the A and C residues of GpApC and of the A and U residues of GpApU are in the low anti range. These features are typical of an A-RNA type structure. In contrast, the G residue of both GpApC and GpApU exists as a mixture of syn and anti conformations. The overall conformation of the two bound ribotrinucleoside bisphosphates is also similar to A-RNA and the stability of the complexes is enhanced by extensive base-base stacking interactions. In addition, it is shown that the binding of the codon GpApC to tRNAAsp induces self-association into a multicomplex system consisting of four GpApC-tRNAAsp complexes, whereas the wobble codon GpApU fails to induce any observable self-association.

Anticodon↗

PubMed1984

Glucose-6-phosphate dehydrogenase. A transferred nuclear Overhauser enhancement study of NADP+ conformations in enzyme-coenzyme binary complexes.

The conformation of NADP+ in glucose-6-phosphate-dehydrogenase--NADP+ binary complexes has been investigated using proton-proton transferred nuclear Overhauser enhancement measurements to determine interproton distance ratios between bound NADP+ protons. The enzymes from Saccharomyces cerevisiae (brewer's yeast and baker's yeast) and Hansenula jadinii (Candida utilis, Torula utilis) form binary complexes with NADP+ in which the glycosidic bond of the adenine moiety is in the anti conformation whereas that of the nicotinamide moiety exists as a syn (69-70%)/anti (30-40%) mixture. The enzymes have similar subunit sizes (Mr approximately 58 000) and it is shown that they bind NADP+ in essentially similar conformations. Inactivation of the baker's yeast enzyme with acetylsalicylic acid caused little if any alteration in the conformation of bound NADP+, and the presence of NADP+ during inactivation afforded very little protection to the enzyme. Inactivation rates were, however, lower in the presence of glucose 6-phosphate. It is concluded that the epsilon-amino group of the lysine residue that is acetylated during the inactivation reaction with acetylsalicylic acid is not necessary for binary complex formation between the enzyme and NADP+, but that it is situated in a part of the molecule affected by formation of the enzyme--glucose-6-phosphate complex. The implication of the findings for the catalytic process, and related evolutionary aspects, are discussed briefly.

Aspirin↗

PubMed1984

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy.

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.

Binding Sites↗

PubMed1984

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study.

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.

Adenosine Triphosphate↗

PubMed1984

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex.

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.

Carbon Isotopes↗

PubMed1984

Interproton distance measurements in solution for a double-stranded DNA undecamer comprising a portion of the specific target site for the cycliC AMP receptor protein in the gal operon. A nuclear Overhauser enhancement study.

Using the proton-proton nuclear Overhauser effect, a large number (150) of interproton distances, both intra- and internucleotide, are determined in solution for a non-self-complementary DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon. It is shown that these distances are very similar to those expected for classical B DNA (RMS difference of 0.5 A) but are significantly different from those expected for classical A DNA (RMS difference 1.1A). Glycosidic (chi) and C4'-C3' (delta) bond torsion angles are obtained by model building on the basis of the intranucleotide interproton distances. Whereas the sugar pucker exhibits little base-to-base variation with delta lying in the range 120 +/- 10 degrees, the glycosidic bond torsion angles of the pyrimidine and purine residues are significantly different, with chi pyr = -120 +/- 10 degrees and chi pur = -90 +/- 10 degrees.

Carrier Proteins↗

PubMed1984

An investigation into the solution structures of two self-complementary DNA oligomers, 5'-d(C-G-T-A-C-G) and 5'-d(A-C-G-C-G-C-G-T), by means of nuclear-Overhauser-enhancement measurements.

A 500 MHz 1H-n.m.r. study on two self-complementary alternating pyrimidine-purine oligodeoxyribonucleotides, 5'-d(C-G-T-A-C-G) and 5'-d(A-C-G-C-G-C-G-T), is presented. By using the proton-proton nuclear Overhauser effect virtually complete assignments are obtained and a large number of interproton distances [113 in the case of 5'-d(C-G-T-A-C-G) and 79 in the case of 5'-d(A-C-G-C-G-C-G-T)], both intra- and inter-nucleotide, are determined. The interproton-distance data are consistent with an overall right-handed B-DNA-type structure for both oligonucleotides, in agreement with their B-type c.d. spectra. However, whereas 5'-d(C-G-T-A-C-G) adopts a conventional B-type structure with a mononucleotide repeating unit, the interproton-distance data provide evidence that 5'-d(A-C-G-C-G-C-G-T) has a dinucleotide repeating unit consisting of alternation in glycosidic bond and sugar pucker conformations.

Base Composition↗

PubMed1984

The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study.

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5'-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3'-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a 'melting' temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3'-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5 X 10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.

Anticodon↗

PubMed1984

Internal mobility in a double-stranded B DNA hexamer and undecamer. A time-dependent proton-proton nuclear Overhauser enhancement study.

The internal mobility of the deoxyribose H2'-H2" and base C(H5)-C(H6) and T(CH3)-T(H6) vectors has been investigated by means of time-dependent nuclear Overhauser enhancement (NOE) measurements in a B DNA hexamer and undecamer. Cross-relaxation rates between these proton pairs are determined from the initial slopes of the time development of the NOEs, and, as the interproton distances between these proton pairs are fixed, apparent correlation times for the 3 interproton vectors are calculated from the cross-relaxation rate data. It is shown that there is little residue to residue variation in the cross-relaxation rates of the interproton vectors within each oligonucleotide, that the mean apparent correlation times of the C(H5)-C(H6) and T(CH3)-T(H6) vectors are approximately equal and significantly greater than that of the H2'-H2" vectors, and that the data for the H2'-H2" vectors of both oligonucleotides and the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer cannot be accounted for by isotropic tumbling alone. The data are analysed in terms of a two motion model with isotropic tumbling and a single internal motion. The relaxation time of the internal motion at 23 degrees C is less than or equal to 1 ns for the H2'-H2" vectors of both oligonucleotides and less than or equal to 3 ns for the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer. In the case of the H2'-H2" vectors, however, the amplitude of the internal motion is found to be too large to be compatible with the known stereochemistry of DNA. This finding can only be explained by invoking additional degrees of internal freedom with a larger number of internal motions of small amplitude of the type deduced from the analysis of crystallographic thermal factors [(1984) J. Mol. Biol. 173, 361-388].

Chemical Phenomena↗

PubMed1984

A nuclear-Overhauser-enhancement study of the solution structure of a double-stranded DNA undecamer comprising a portion of the specific target site for the cyclic-AMP-receptor protein in the gal operon. Sequential resonance assignment.

A 500-MHz 1H-NMR study on a double-stranded non-self-complementary DNA undecamer comprising a portion of the specific target site for the cyclic AMP receptor protein in the gal operon is presented. Using pre-steady-state nuclear Overhauser effect (NOE) measurements, all exchangeable imino, non-exchangeable base, methyl, and H1', H2' and H2" sugar proton resonances are assigned in a sequential manner. In addition, some of the H3' sugar proton resonances are also assigned and some of the exchangeable amino proton resonances identified. The relative magnitudes of the intranucleotide and internucleotide NOEs are indicative of a right-handed B-type conformation for the duplex undecamer in solution.

Base Sequence↗

PubMed1984

Structure of the ribotrinucleoside diphosphate codon UpUpC bound to tRNAPhe from yeast. A time-dependent transferred nuclear Overhauser enhancement study.

The structure of the ribotrinucleoside diphosphate UpUpC, the codon for phenylalanine, bound to yeast tRNAPhe in solution is elucidated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The glycosidic bond and ribose conformations are low anti and 3'-endo, respectively, typical of an A-RNA type structure. The main chain torsion angles are all within the range of those expected for A-RNA but small differences from those in conventional A-RNA 11 result in a special structure with a larger rotation per residue (40 to 45 degrees compared to 32.7 degrees in R-RNA 11) and almost perfect stacking of the bases. These two structural features, which are similar to those found in the anticodon triplet of the monoclinic crystal form of tRNAPhe, can account for the known greater stability of the codon-anticodon complex relative to an equivalent double helical RNA trimer with a conventional A-RNA structure.

Codon↗

PubMed1984

An unusual conformation of NAD+ bound to sorbitol dehydrogenase? A time-dependent transferred nuclear Overhauser effect study.

The conformation of NAD+ in the sheep liver sorbitol dehydrogenase-NAD+ binary complex has been investigated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine interproton distance ratios and distances between bound NAD+ protons. The conformation about both the adenosine and nicotinamide riboside glycosidic bonds is anti, the conformations of the adenosine and nicotinamide ribose rings are C3'-endo and C1'-exo, respectively, and the conformations about the adenosine and nicotinamide riboside C4'-C5' bonds are g+ and t, respectively, similar to those found in complexes of NAD+ with other A type dehydrogenases. In addition, however, the distance data are indicative of an unusual overall conformation of NAD+ in the sorbitol dehydrogenase-NAD+ binary complex, with the planes of the nicotinamide and adenine rings separated by 6 to 8 A and at approximately 120 degrees to each other. This overall conformation differs from the concensus extended conformation found in the NAD+-dehydrogenase complexes crystallized to date, where the planes of the nicotinamide and adenine rings are 12 to 14 A apart and nearly perpendicular to each other.

Animals↗