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G M Clore

Publications and source records attributed to G M Clore.

At least 163 records · Page 9Linked to original sources
PubMed1991

Comparison of the solution nuclear magnetic resonance and X-ray crystal structures of human recombinant interleukin-1 beta.

The solution structure of interleukin-1 beta determined by nuclear magnetic resonance spectroscopy is compared to three independently solved X-ray structures at 2 A resolution. It is shown that the solution and X-ray structures are very similar, both locally and globally. The atomic root-mean-square (r.m.s.) difference between the solution and X-ray structures is approximately 0.9 A for backbone atoms, approximately 1.5 A for all atoms and approximately 1 A for all atoms of internal residues. The largest differences are confined to some of the loops and turns connecting beta-strands. The atomic r.m.s. distribution of the 32 calculated solution structures about their mean co-ordinate positions (approximately 0.4 A for backbone atoms, approximately 0.8 A for all atoms and approximately 0.5 A for all atoms of internal residues) is approximately the same as the atomic r.m.s. differences between the three X-ray structures, indicating that the positional errors in the atomic co-ordinates determined by the two methods are similar.

Humans↗
PubMed1991

A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G.

The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (A) for the backbone atoms, 0.65 A for all atoms, and 0.39 A for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded beta sheet, on top of which lies a long helix. The central two strands (beta 1 and beta 4), comprising the NH2- and COOH-termini, are parallel, and the outer two strands (beta 2 and beta 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87 degrees C).

Amino Acid Sequence↗
PubMed1991

Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

The presence of bound water molecules in the solution structure of reduced human thioredoxin has been investigated using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. It is demonstrated that the backbone amide protons of Lys21, Lys39, Lys82, Gly83 and Asn102, as well as the side-chain amide group of Asn102, are in close proximity to bound water molecules. Examination of the high-resolution solution structure of reduced human thioredoxin reveals that these results are best accounted for by four bound water molecules. Subsequent simulated annealing calculations carried out on the basis of interproton distance and hydrogen bonding restraints to the bound water molecules, supplemented by the original set of experimental restraints used in the calculation of the three-dimensional structure of human thioredoxin, permit a more precise localization of the bound water positions. Potential hydrogen bonds to these water molecules are described and a comparison is made to corresponding bound water molecules in the crystal structure of oxidized Escherichia coli thioredoxin.

Amino Acid Sequence↗
PubMed1991

Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy.

Three- and four-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy offers dramatic improvements in spectral resolution by spreading through-bond and through-space correlations in three and four orthogonal frequency axes. Simultaneously, large heteronuclear couplings are exploited to circumvent problems due to the larger linewidths that are associated with increasing molecular weight. These novel experiments have been designed to extend the application of NMR as a method for determining three-dimensional structures of proteins in solution beyond the limits of conventional two-dimensional NMR (approximately 100 residues) to molecules in the 150- to 300-residue range. This potential has recently been confirmed with the determination of the high-resolution NMR structure of a protein greater than 150 residues, namely, interleukin-1 beta.

Interleukin-1↗
PubMed1991

Specific DNA binding to a major histocompatibility complex enhancer sequence by a synthetic 57-residue double zinc finger peptide from a human enhancer binding protein.

Two 57-residue peptides containing one pair of "zinc fingers" from a human enhancer binding protein were prepared by solid-phase peptide synthesis. One peptide (MBP-DF) contained the native sequence, while the second peptide ([Abu11]MBP-DF) has an alpha-aminobutyric acid residue substituted for a nonconserved cysteine residue at position 11. The peptides were characterized by several chemical and physical methods, and their DNA binding properties were evaluated using gel retardation experiments. Spectroscopic studies demonstrated that addition of metal ions such as zinc and cobalt resulted in specific conformational changes in both peptides, indicating that cysteine-11 does not appear to be involved in metal chelation. One-dimensional 1H NMR studies indicate that a stable folded structure is formed upon addition of zinc, and the chemical shift pattern is consistent with that previously observed for one constituent single finger (Omichinski, J., Clore, G. M., Appella, E., Sakaguchi, K., and Gronenborn, A. M. (1990) Biochemistry 29, 9324-9334). Gel retardation experiments demonstrate that the peptides are capable of interacting with a 15-mer oligonucleotide comprising a portion of the major histocompatibility complex enhancer sequence and that the interaction is zinc-dependent. The dissociation constant for the [Abu11]MBP-DF peptide is 1.4 x 10(-7) M with maximal binding occurring at a zinc-to-peptide ratio of 2 to 1. The binding specificity observed with respect to related enhancer sequences exhibits the same relative order as noted previously for the whole protein. Studies with point mutants of the major histocompatibility complex enhancer binding sequence indicate that the last GC base pair in a four-guanine stretch plays a pivotal role in the interaction between the peptide and DNA.

Amino Acid Sequence↗
PubMed1991

High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution.

The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements. In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants. The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints. The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms. The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines. The overall structure is similar to the crystal and NMR structures of oxidized [Katti, S. K., LeMaster, D. M., & Eklund, H. (1990) J. Mol. Biol. 212, 167-184] and reduced [Dyson, J. H., Gippert, G. P., Case, D. A., Holmgren, A., & Wright, P. (1990) Biochemistry 29, 4129-4136] Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology. Several differences, however, can be noted. The human alpha 1 helix is a full turn longer than the corresponding helix in E. coli thioredoxin and is characterized by a more regular helical geometry. The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E. coli protein and is also longer in the human protein. In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.

Amino Acid Sequence↗
PubMed1991

High-resolution three-dimensional structure of interleukin 1 beta in solution by three- and four-dimensional nuclear magnetic resonance spectroscopy.

The determination of the high-resolution three-dimensional solution structure of interleukin 1 beta (IL-1 beta), a protein of 153 residues and 17.4 kDa, which plays a central role in the immune and inflammatory responses, has been determined by heteronuclear (13C and 15N) three- and four-dimensional NMR spectroscopy. The structure is based on 3146 experimental restraints comprising 2780 distance and 366 torsion angle (phi, psi, and chi 1) restraints. A total of 32 simulated annealing structures are calculated, and the atomic RMS distribution about the mean coordinate positions is 0.41 +/- 0.04 A for the backbone atoms and 0.82 +/- 0.04 A for all atoms (excluding residue 1 at the N-terminus and residues 152 and 153 at the C-terminus, which are partially disordered). In the case of internal side chains with a surface accessibility of less than or equal to 40%, the atomic RMS distribution about the mean coordinate positions for all atoms is 0.49 +/- 0.03 A. IL-1 beta resembles a tetrahedron and is composed of 12 beta-strands arranged in three pseudosymmetric topological units, each of which comprises 5 strands. Analysis of the mutational data on IL-1 beta in the light of the three-dimensional structure suggests the presence of three distinct binding sites for the IL-1 receptor on the surface of the protein. It is suggested that each of the three immunoglobulin domains which comprise the extracellular portion of the IL-1 receptor recognizes one of these sites.

Amino Acid Sequence↗
PubMed1991

Comparison of the solution nuclear magnetic resonance and crystal structures of interleukin-8. Possible implications for the mechanism of receptor binding.

A comparison of the solution structure of the interleukin-8 dimer determined by nuclear magnetic resonance spectroscopy with that of the 2 A resolution X-ray structure, solved by molecular replacement using the solution structure as a starting model, is presented. At the monomer level the atomic root-mean-square difference between the two structures for residues 7 to 72 is approximately 1.1 A for the backbone atoms, approximately 1.6 A for all atoms, and approximately 1 A for all atoms of the internal residues. There are two main regions of difference in the monomer. In the X-ray structure residues 4 to 6 are well ordered and the charged groups of Glu4 of one subunit and Lys23' of the other are in close enough proximity to form an electrostatic interaction. In contrast, these residues are partially disordered in solution and the electrostatic interaction involving Glu4 is replaced by one between Glu29 of one subunit and Lys23' of the other. In the loop comprising residues 31 to 36, His33 accepts a hydrogen bond from the backbone amide group of Gln8 in the solution structure, but donates a hydrogen bond to the backbone carbonyl group of Glu29 in the X-ray structure. There is also a difference in the quaternary structure with regard to the relative orientation of the two subunits produced by a rigid body rotation about the C2 axis that alters the angle between the central beta-strands (formed by residues 23 to 29 of the 2 subunits) at the dimer interface, without breaking the symmetry. In the solution structure this angle has a value of 168 degrees, while in the X-ray structure the central strands are essentially flat, with an angle of 179 degrees. As a result, the separation between the two anti-parallel helices, which lie at an angle of about 60 degrees to the underlying beta-strands, is decreased from 14.8 A in the solution structure to 11.1 A in the X-ray structure. The quaternary structural difference is related to the different conformations of the N terminus and the 31 to 36 loop, both of which display different interactions with respect to the ends of the central beta-strands in the two structures. These findings indicate that interleukin-8 has the potential to undergo conformational transitions that may be of functional significance.

Hydrogen Bonding↗
PubMed1991

Crystal structure of interleukin 8: symbiosis of NMR and crystallography.

The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement using as a model the solution structure derived from nuclear magnetic resonance experiments. The structure was refined with 2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A). A comparison indicates some potential differences between the structure in solution and in the crystalline state. Our analysis also predicts that residues 4 through 9 on the amino terminus and the beta-bend, which includes His-33, may be important for receptor binding.

Amino Acid Sequence↗
PubMed1991

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta.

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗
PubMed1991

Stereospecific assignment of beta-methylene protons in larger proteins using 3D 15N-separated Hartmann-Hahn and 13C-separated rotating frame Overhauser spectroscopy.

3J alpha beta coupling constants and complementary nuclear Overhauser data on the intraresidue C alpha H-C beta H distances form an essential part of the data needed to obtain stereospecific assignments of beta-methylene protons in proteins. In this paper we show that information regarding the magnitude of the 3J alpha beta coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D 15N-separated 1H-1H Hartmann-Hahn 1H-15N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C alpha H-C beta H distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D 13C-separated 1H-1H rotating frame Overhauser effect 1H-13C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1 beta, a protein of 153 residues and 17.4 kDa molecular weight.

Amino Acids↗
PubMed1991

Modeling the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 on the basis of the solution structure of interleukin-8.

A model of the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 is presented. The model is predicted based on the previously determined solution structure of interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry 29, 1689-1696]. Both proteins belong to a superfamily of cytokine proteins involved in cell-specific chemotaxis, host defense and the inflammatory response. The amino acid sequence identity between the two proteins is 24%. It is shown that the regular secondary structure elements of the parent structure can be retained in the modeled structure, such that the backbone hydrogen bonding pattern is very similar in the two structures. The polypeptide backbone is superimposable with an atomic r.m.s. difference of 0.9 A and all side chains can be modeled by transferring the parent side chain conformation to the new structure. Thus, the deduced structure, like the parent one, is a dimer and consists of a six-stranded antiparallel beta-sheet, formed by two three-stranded Greek keys, one from each monomer, upon which lie two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by approximately 14 A. All amino acid sequence changes can be accommodated within the parent polypeptide framework without major rearrangements. This is borne out by the fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structures have similar non-bonding energies. These results strongly suggest that both proteins and all other members of the superfamily most likely have the same tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

High-resolution three-dimensional structure of a single zinc finger from a human enhancer binding protein in solution.

The three-dimensional structure of a 30-residue synthetic peptide containing the carboxy-terminal "zinc finger" motif of a human enhancer binding protein has been determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on 487 approximate interproton distance and 63 torsion angle (phi, psi, and chi 1) restraints. A total of 40 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 29 and 30 which are ill-defined) is 0.4 A for the backbone atoms, 0.8 A for all atoms, and 0.41 A for all atoms excluding the lysine and arginine side chains, which are disordered. The solution structure of the zinc finger consists of two irregular antiparallel beta-strands connected by an atypical turn (residues 3-12) and a classical alpha-helix (residues 14-24). The zinc is tetrahedrally coordinated to the sulfur atoms of two cysteines (Cys-5 and Cys-8) and to the N epsilon 2 atoms of two histidines (His-21 and His-27). The two cysteine residues are located in the turn connecting the two beta-strands (residues 5-8); one of the histidine ligands (His-21) is in the alpha-helix, while the second histidine (His-27) is at the end of a looplike structure (formed by the end of the alpha-helix and a turn). The general architecture is qualitatively similar to two previously determined low-resolution Cys2-His2 zinc finger structures, although distinct differences can be observed in the beta-strands and turn and in the region around the two histidines coordinated to zinc. Comparison of the overall polypeptide fold of the enhancer binding protein zinc finger with known structures in the crystallographic data base reveals a striking similarity to one region (residues 23-44) of the X-ray structure of proteinase inhibitor domain III of Japanese quail ovomucoid [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I., & Laskowski, M. (1982) J. Mol. Biol. 158, 515-537], which could be superimposed with a backbone atomic rms difference of 0.95 A on residues 3-25 (excluding residue 6) of the zinc finger from the enhancer binding protein. The presence of structural homology between two proteins of very different function may indicate that the so-called zinc finger motif is not unique for a class of DNA binding proteins but may represent a general folding motif found in a variety of proteins irrespective of their function.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli.

The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the lambda PL promoter. The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion-exchange and size exclusion chromatography. The purified protein appears to be in a native-like homogeneous conformational state as determined by 1H-NMR spectroscopy and circular dichroism measurements. HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441.

Amino Acid Sequence↗
PubMed1990

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Isotopes↗
PubMed1990

Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.

Hydrogen↗