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G M Clore

Publications and source records attributed to G M Clore.

At least 127 records · Page 7Linked to original sources
PubMed1994

Fast folding of a prototypic polypeptide: the immunoglobulin binding domain of streptococcal protein G.

The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched-flow deuterium-hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND-NH exchange up to 10-fold higher than that expected for a random coil, occurs within the dead time (< or = 1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half-life of 5.2 ms at 5 degrees C.

Bacterial Proteins↗
PubMed1994

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.

Amino Acid Sequence↗
PubMed1994

Identification of N-terminal helix capping boxes by means of 13C chemical shifts.

We have examined the 13C alpha and 13C beta chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with phi and psi clustering around 94 +/- 15 degrees and 167 +/- 5 degrees, respectively. This is manifested by a 1-2 ppm upfield shift of the 13 C alpha resonance and a 1-4 ppm downfield shift of the 13C beta resonance, relative to their random coil values, and is mainly associated with the unusually large value of psi. The residues following the N-cap residue exhibit downfield shifts of 1-3 ppm for the 13C alpha resonances and small upfield shifts for the 13C beta ones, typical of an alpha-helix.

Amino Acid Sequence↗
PubMed1994

Comparison of four independently determined structures of human recombinant interleukin-4.

Four independent structures of human interleukin-4, two determined by nuclear magnetic resonance techniques and two by X-ray diffraction, have been compared in detail. The core of this four helix bundle protein is very similar in all the structures but there are some differences in loop regions that are known to be mobile in solution. Careful comparison of the experimental data sets and the methods of analysis of the different laboratories has provided clues to the sources of most of the differences, and also answered some general questions about the accuracy of protein structure determination by these two techniques.

Crystallography, X-Ray↗
PubMed1993

Identification of the contact surface of a streptococcal protein G domain complexed with a human Fc fragment.

The B1 domain of streptococcal protein G interacts with the C-terminal fragment of the heavy chain of immunoglobulin G (IgGFc). The binding site for the protein G domain on the antibody fragment is in close proximity or overlapping with that determined for staphylococcal protein A. The interaction of the B1 domain with IgGFc was investigated by 1H-15N correlation spectroscopy. The major interaction site on the B1 domain comprises parts of beta-strand 3 as well as the alpha-helix. Comparison with the crystal structure of the protein A/IgGFc complex suggests that the mode of interaction in the two complexes is analogous, despite the lack of sequence or structural similarity between two antibody binding proteins.

Bacterial Proteins↗
PubMed1993

NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1.

The three-dimensional solution structure of a complex between the DNA binding domain of the chicken erythroid transcription factor GATA-1 and its cognate DNA site has been determined with multidimensional heteronuclear magnetic resonance spectroscopy. The DNA binding domain consists of a core which contains a zinc coordinated by four cysteines and a carboxyl-terminal tail. The core is composed of two irregular antiparallel beta sheets and an alpha helix, followed by a long loop that leads into the carboxyl-terminal tail. The amino-terminal part of the core, including the helix, is similar in structure, although not in sequence, to the amino-terminal zinc module of the glucocorticoid receptor DNA binding domain. In the other regions, the structures of these two DNA binding domains are entirely different. The DNA target site in contact with the protein spans eight base pairs. The helix and the loop connecting the two antiparallel beta sheets interact with the major groove of the DNA. The carboxyl-terminal tail, which is an essential determinant of specific binding, wraps around into the minor groove. The complex resembles a hand holding a rope with the palm and fingers representing the protein core and the thumb, the carboxyl-terminal tail. The specific interactions between GATA-1 and DNA in the major groove are mainly hydrophobic in nature, which accounts for the preponderance of thymines in the target site. A large number of interactions are observed with the phosphate backbone.

Amino Acid Sequence↗
PubMed1993

Assessing the quality of solution nuclear magnetic resonance structures by complete cross-validation.

Structure determination of macromolecules in solution by nuclear magnetic resonance (NMR) spectroscopy involves the fitting of atomic models to the observed nuclear Overhauser effect (NOE) data. Complete cross-validation has been used to define reliable and unbiased criteria for the quality of solution NMR structures. The method is based on the partitioning of NOE data into test sets and the cross-validation of statistical quantities for each of the test sets. A high correlation between cross-validated measures of fit, such as distance bound violations and NMR R values, and the quality of solution NMR structures was observed. Less complete data resulted in poorer satisfaction of the cross-validated measures of fit. Optimization of cross-validated measures of fit will likely produce solution NMR structures with maximal information content.

Bacterial Proteins↗
PubMed1993

The high-resolution, three-dimensional solution structure of human interleukin-4 determined by multidimensional heteronuclear magnetic resonance spectroscopy.

The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4), a protein of approximately 15 kDa which plays a key role in the regulation of B and T lymphocytes, has been determined using three- and four-dimensional heteronuclear NMR spectroscopy. The structure is based on a total of 2973 experimental NMR restraints, comprising 2515 approximate interproton distance restraints, 102 distance restraints for 51 backbone hydrogen bonds, and 356 torsion angle restraints. A total of 30 structures was calculated by means of hybrid distance geometry-simulated annealing, and the atomic rms distribution about the mean coordinate positions for residues 8-129 is 0.44 +/- 0.03 A for the backbone atoms, 0.83 +/- 0.03 A for all atoms, and 0.51 +/- 0.04 A for all atoms excluding disordered side chains. The N- and C-terminal residues (1-7 and 130-133, respectively) appear to be disordered. The structure of IL-4 is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections. The linker elements between the helices are formed by either long loops, small helical turns, or short strands. The latter include a mini anti-parallel beta-sheet. A best fit superposition of the NMR structure of IL-4 with the 2.25 A resolution crystal structure [Wlodawer, A., Pavlovsky, A., & Gutschina, A. (1992) FEBS Lett. 309, 59-64] yields a backbone atomic rms difference of 1.37 A which can be mainly attributed to tighter packing of the helices in the crystal structure. This is indicated by an approximately 20% reduction in the axial separation of three pairs of helices (alpha A-alpha C, alpha A-alpha D, and alpha C-alpha D) in the crystal structure relative to the NMR structure and may reflect the greater flexibility of the molecule in solution which is reduced in the crystal due to intermolecular contacts. Comparison of the NMR structure of IL-4 with the X-ray structures of two other related proteins, granulocyte-macrophage colony stimulating factor [Diedrichs, K., Boone, T., & Karplus, P. A. (1992) Science 254, 1779-1782] and human growth hormone [de Vos, A. M., Ultsch, M., & Kossiakoff, A. A. (1992) Science 255, 306-312], that bind to the same hematopoietic superfamily of cell surface receptors reveals a remarkably similar topological fold, despite the absence of any significant overall sequence identity, and substantial differences in the relative lengths of the helices, the lengths and the nature of the various connecting elements, and the pattern and number of disulfide bridges.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.

The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.

Base Sequence↗
PubMed1993

Kinetics of folding of the all-beta sheet protein interleukin-1 beta.

The folding of the all-beta sheet protein, interleukin-1 beta, was studied with nuclear magnetic resonance (NMR) spectroscopy, circular dichroism, and fluorescence. Ninety percent of the beta structure present in the native protein, as monitored by far-ultraviolet circular dichroism, was attained within 25 milliseconds, correlating with the first kinetic phase determined by tryptophan and 1-anilinonaphthalene-8-sulfonate fluorescence. In contrast, formation of stable native secondary structure, as measured by quenched-flow deuterium-hydrogen exchange experiments, began after only 1 second. Results from the NMR experiments indicated the formation of at least two intermediates with half-lives of 0.7 to 1.5 and 15 to 25 seconds. The final stabilization of the secondary structure, however, occurs on a time scale much greater than 25 seconds. These results differ from previous results on mixed alpha helix-beta sheet proteins in which both the alpha helices and beta sheets were stabilized very rapidly (less than 10 to 20 milliseconds).

Circular Dichroism↗
PubMed1993

Exploring the limits of precision and accuracy of protein structures determined by nuclear magnetic resonance spectroscopy.

The effects of the number, precision and accuracy of interproton distance restraints, of direct refinement against nuclear Overhauser enhancement (NOE) intensities and of the description of the non-bonded contacts on the precision and accuracy of a nuclear magnetic resonance (NMR) protein structure determination have been investigated. The model system employed is the 56 residue immunoglobulin G binding domain of streptococcal protein G. This choice was based on the availability of a very high resolution NMR structure (atomic root-mean-square distribution of the ensemble of 60 calculated structures about the mean co-ordinate positions of 0.25 A for the backbone atoms, 0.65 A for all atoms and 0.39 A for all atoms excluding disordered surface side-chains). The experimental NMR data set for this structure determination comprised a total of 1058 experimental restraints of which 854 were approximate interproton distance restraints corresponding to all the structurally useful NOEs observable for this protein. The calculations presented in this paper reveal the following. (1) The number of interproton distance restraints constitutes the single most important determinant of both precision and accuracy. The ensemble precision and accuracy improves significantly as the number of interproton distance restraints is increased to an average of approximately 15 per residue, of which approximately 60% involve unique proton pairs; subsequent additions of interproton distance restraints, however, lead to less dramatic improvements as information redundancy sets in. (2) The ratio of ensemble precision to ensemble accuracy (which ranges from 0.5 to 0.7 for the backbone atoms) is approximately independent both of the number, precision and accuracy of the interproton distance restraints, and of whether the structures are refined against interproton distance restraints or directly against NOE intensities. (3) In an ensemble of structures generated from a large number of loose approximate interproton distance restraints (an average of approximately 15 restraints per residue with approximately 60% involving unique proton pairs), the interproton distance vectors corresponding to the restraints are very well defined with approximately 80% of vectors between unique proton pairs having a standard deviation of < or = 0.1 A. (4) The accuracy of the mean co-ordinates of an ensemble of structures is significantly higher than the average accuracy of the individual structures comprising the ensemble. For an average ensemble precision of > or = 0.6 A, the dependence of the accuracy of the mean co-ordinates on ensemble precision is approximately linear.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1993

Analysis of the backbone dynamics of interleukin-8 by 15N relaxation measurements.

The backbone dynamics of the cytokine interleukin-8, a symmetric homodimer of overall molecular mass 16 kDa, has been investigated at pH 5.2 by means of 15N relaxation measurements using heteronuclear two-dimensional inverse detected 1H-15N spectroscopy. 15N T1, T2 and NOE data were obtained for 66 out of a total of 67 backbone amide groups. The overall correlation time is 9.10(+/- 0.05) ns at 26.6 degrees C. All residues exhibit very rapid motions on a time-scale of < or = 20 ps. These very rapid motions alone can account for the 15N relaxation behaviour of 30 residues. The 15N relaxation data for another 21 residues can only be accounted for by the inclusion of an additional internal motion on a time-scale ranging from 0.5 to 3.5 ns. These residues are clustered at the N and C termini, and in the loop regions connecting elements of secondary structures. Finally, the 15N relaxation data for another 15 residues could only be accounted for by the presence of chemical exchange on a time-scale ranging from approximately 170 ns to 2.25 ms. In addition, the inclusion of chemical exchange improved the fit to the experimental data for 10 of the 30 residues whose 15N relaxation behaviour could be accounted for by very fast motions alone. The residues exhibiting chemical exchange line broadening cluster at the interface of the long C-terminal alpha-helix and the underlying beta-sheet. It is suggested that this clustering is indicative of concerted rather than independent motions in regions of secondary structure, with motion at any one residue being propagated to neighbouring residues in van der Waals contact.

Hydrogen-Ion Concentration↗
PubMed1993

A small single-"finger" peptide from the erythroid transcription factor GATA-1 binds specifically to DNA as a zinc or iron complex.

Sequence-specific DNA binding has been demonstrated for a synthetic peptide comprising only one of the two "finger"-like domains of the erythroid transcription factor GATA-1 (also termed Eryf-1, NF-E1, or GF-1). Quantitative analysis of gel-retardation assays yields a specific association constant of 1.2 x 10(8) M, compared with values of about 10(9) M for the full-length natural GATA-1 protein. By the use of peptides of various lengths, it was possible to delineate the smallest region necessary for specific binding. A single C-terminal finger of the double-finger motif is necessary but not sufficient for sequence-specific interaction. Basic amino acids located C-terminal to the finger (some more than 20 amino acids away) are also essential for tight binding. In addition to demonstrating that zinc is important for the formation of an active binding complex, we show that other ions, notably Fe2+, can fulfill this role. Our results make it clear that the GATA-1 metal binding motif is quite distinct from that found in the steroid hormone family and that GATA-1 is a member of a separate class of DNA binding proteins.

Amino Acid Sequence↗