PubMed Health⌕ Search

Biomedical subjects

M Rance

Publications and source records attributed to M Rance.

18 recordsLinked to original sources
PubMed1996

Some practical aspects of double-resonance techniques in solution-state NMR studies of high-molecular-weight systems.

This paper addresses some of the practical issues associated with solution NMR investigations of high-molecular-weight ( > 20 kDa) biomolecules and their complexes. The experimental example of a high-molecular-weight system used here is a 52 kDa Fab/peptide complex. Theoretical as well as experimental aspects of solution NMR techniques employed to study such larger systems are discussed. Specific topics covered include hardware considerations, solvent suppression, and the relative efficiencies of various isotope-edited NMR techniques. Typical NOE methods used to derive structural information on these higher-molecular-weight systems and the scope of structural detail achievable are also examined.

Antibodies, Monoclonal↗
PubMed1992

Two-dimensional NMR studies of d(GGTTAATGCGGT).d(ACCGCATTAACC) complexed with the minor groove binding drug SN-6999.

The dodecadeoxynucleotide duplex d(GGTTAATGCGGT).d(ACCGCATTAACC) and its 1:1 complex with the minor groove binding drug SN-6999 have been prepared and studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments have been obtained for the free duplex by standard methods. The line widths of the resonances in the complex are greater than those observed for the free duplex, which complicates the assignment process. Extensive use of two-quantum spectroscopy was required to determine the scalar correlations for identifying all of the base proton and most of the 1'H-2'H-2''H spin subsystems for the complex. This permitted unambiguous sequence-specific resonance assignments for the complex, which provides the necessary background for a detailed comparison of the structure of the duplex, with and without bound drug. A series of intermolecular NOEs between drug and DNA were identified, providing sufficient structural constraints to position the drug in the minor groove of the duplex. However, the combination of NOEs observed can only be rationalized by a model wherein the drug binds in the minor groove of the DNA in both orientations relative to the long helix axis and exchanges rapidly between the two orientations. The drug binds primarily in the segment of five consecutive dA-dT base pairs d(T3T4A5A6T7).d(A18T19T20A21A22), but surprisingly strong interactions are found to extend one residue in the 3' direction along each strand to G8 and C23. The observation of intermolecular contacts to residues neighboring the AT-rich region demonstrates that the stabilization of the bis(quaternary ammonium) heterocycle family of AT-specific, minor groove binding drugs is not based exclusively on interactions with dA-dT base pairs.

Antineoplastic Agents↗
PubMed1992

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].

Amino Acid Sequence↗
PubMed1992

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy.

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.

Amino Acid Sequence↗
PubMed1991

Comparative NMR analysis of the decadeoxynucleotide d-(GCATTAATGC)2 and an analogue containing 2-aminoadenine.

The dodecadeoxynucleotide duplex d-(GCATTAATGC)2 has been prepared with all adenine bases replaced by 2-NH2-adenine. This modified duplex has been characterized by nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific 1H resonance assignments have been obtained by using a variety of 2D NMR methods. Multiple quantum-filtered and multiple quantum experiments have been used to completely assign all sugar ring protons, including 5'H and 5'H resonances. The assignments form the basis for a detailed comparative analysis of the 1H NMR parameters of the modified and parent duplex. The structural features of both decamer duplexes in solution are characteristic of the B-DNA family. The spin-spin coupling constants in the sugar rings and the relative spatial proximities of protons in the bases and sugars (as determined from the comparison of corresponding nuclear Overhauser effects) are virtually identical in the parent and modified duplexes. Thus, substitution by this adenine analogue in oligonucleotides appears not to disturb the global or local conformation of the DNA duplex.

2-Aminopurine↗
PubMed1988

Complete assignment of the 1H nuclear magnetic resonance spectrum of French bean plastocyanin. Application of an integrated approach to spin system identification in proteins.

The identification of the spin systems that comprise the 1H nuclear magnetic resonance spectrum of French bean Cu(I) plastocyanin (Mr 10,600) has been made using an approach that integrates a wide range of two-dimensional nuclear magnetic resonance experiments. A very large percentage of these assignments has been obtained in spectra acquired from 1H2O solution using a backbone amide-based strategy. The spin systems of 91 of the 99 residues have been assigned to the appropriate amino acid, thereby providing an ample basis for obtaining sequence-specific assignments, as described in the accompanying paper.

Amino Acid Sequence↗
PubMed1988

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn.

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1988

Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix.

1H nuclear magnetic resonance experiments indicate formation of secondary structures in water solutions of a synthetic immunogenic peptide of sequence EVVPHKKMHKDFLEKIGGL corresponding to the C-helix (residues 69 to 87) of myohemerythrin. The conformational ensemble consists of a set of turn-like structures, distributed over the C-terminal half of the peptide and rapidly interconverting by way of unfolded states. These structures, termed nascent helix, are stabilized into helical structure with long-range order in water/trifluorethanol mixtures. Circular dichroism measurements confirm the presence of 50% helix in water/trifluoroethanol but show no evidence of helicity in water solutions of the peptide. It is apparent that no one member of the transient set of helical conformations which constitutes the nascent helix is sufficiently long to be detectable by circular dichroism experiments. No preferred conformations could be detected by nuclear magnetic resonance in the N-terminal half of the peptide, either in water or water/trifluoroethanol mixtures. This region of the peptide is stabilized in helix by long-range interactions in the folded protein. The possible role of nascent secondary structure in induction of antipeptide antibodies and in initiation of protein folding is discussed.

Amino Acid Sequence↗
PubMed1987

Complete assignment of lysine resonances in 1H NMR spectra of proteins as probes of surface structure and dynamics.

A strategy is presented for complete identification of 1H spin systems of lysine residues using sophisticated 2D NMR experiments. Relayed and remote connectivities within each spin system are determined for spin subsystems based at the backbone amide and C epsilon proton resonances. When complete spin system identification is combined with sequence-specific assignment, protein surface structure and dynamics can be probed in a site-specific manner. The interaction between the five lysine residues of French bean plastocyanin and a model redox partner Cr(CN)6(3-) has been examined using this approach.

Lysine↗
PubMed1986

1H nuclear magnetic resonance assignments for d-(GCATTAATGC)2 using experimental refinements of established procedures.

Sequence-specific 1H nuclear magnetic resonance assignments are presented for d-(GCATTAATGC)2. Using omega 1-scaled double quantum-filtered correlated spectroscopy, two-quantum spectroscopy, relayed coherence transfer spectroscopy and detailed analysis of the fine structure in these phase-sensitive spectra, the spin system of the bases and deoxyribose rings were identified entirely via scalar proton-proton couplings. The sequential connectivities were established with two-dimensional nuclear Overhauser enhancement spectra recorded with a short mixing time of 60 milliseconds. These spectra contain only a small number of cross-peaks, corresponding to the shortest proton-proton distances prevailing in the DNA. They are thus easy to interpret, and therefore the presently proposed modifications of the established assignment procedures should enable studies of larger DNA duplexes with intrinsically more complex nuclear magnetic resonance spectra, and they also provided an improved basis for conformational studies of DNA fragments.

DNA↗
PubMed1985

Simplification of 1H NMR spectra of proteins by one-dimensional multiple quantum filtration.

The use of multiple quantum filters for simplification and editing of one-dimensional 1H NMR spectra of proteins is demonstrated. Three, four and five quantum-filtered spectra have been recorded. The technique is applicable to proteins of molecular weight up to at least 18,000. Examples obtained for a mixture of amino acids and for the proteins plastocyanin and myoglobin are reported. A remarkable degree of spectral editing can be achieved by judicious choice of experimental parameters.

Amino Acids↗
PubMed1983

Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering.

A double quantum filter is inserted into a two-dimensional correlated (COSY) 1H NMR experiment to obtain phase-sensitive spectra in which both cross peak and diagonal peak multiplets have anti-phase fine structure, and in which the cross peaks and the major contribution to the diagonal peaks have absorption lineshapes in both dimensions. The elimination of the dispersive character of the diagonal peaks in phase-sensitive, double quantum-filtered COSY spectra allows identification of cross peaks lying immediately adjacent to the diagonal, which represents a significant improvement over the conventional COSY experiment.

Magnetic Resonance Spectroscopy↗
PubMed1982

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of acholeplasma laidlawii. A 2H-NMR study.

We have investigated by 2H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of acholeplasma laidlawii B. This is the only 2H-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristic condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the 2H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.

Acholeplasma laidlawii↗
PubMed1980

Orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii as observed by 2H-NMR.

Oleic acid specifically deuterated at fifteen different positions along the chain, including the double bond, was biosynthetically incorporated into the membrane lipids of the microorganism Acholeplasma laidlawii B. A detailed study of the dynamic conformation of these chains was carried out using deuterium nuclear magnetic resonance. The deuterium spectra fourteen different samples were recorded as a function of temperature over the range 0-41 degrees C. Spectra were obtained down to -52 degrees C for the sample enriched with oleic acid deuterated at the C-12' position. Above 20 degrees C, where the lipids are in the liquid crystal phase, a single quadrupolar powder pattern was observed for each C2H2 segment, except for the C-2' position which gave rise to a three-component spectrum characteristic for this position in both model and biological membranes. Simulation of this spectrum indicates that there are two conformations of the lipid molecule in the region of the C-2' segment of the sn-2 chain. The orientationa fluctuations of the fatty acid chain segments in the A. laidlawii membranes are described by the deuterium order parameters, and a striking similarity is shown to exist between the oleate chain conformation of the A. laidlawii membrane and a phospholipid model membrane. Remarkable similarities are also demonstrated in the A. laidlawii membrane enriched in palmitic and oleic fatty acids when the order parameter profiles are plotted at the same reduced temperature. Below 15 degrees C a second component, due to gel phase lipid, starts to appear in the spectra. This broad gel phase component grows at the expense of the liquid crystal phase component as the temperature is reduced. The spectra indicate that the center of the phase transition is at about -12 degrees C, in good agreement with DSC studies.

Acholeplasma laidlawii↗