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G M Clore

Publications and source records attributed to G M Clore.

At least 109 records · Page 6Linked to original sources
PubMed1995

Solvent isotope effect and protein stability.

Here we present a comparative study of the stability of several proteins in H2O and D2O as a function of pH/pH*. We show that the substitution of D2O for H2O leads to an increase in the transition temperature and a decrease in the enthalpy of unfolding. The stability of the proteins, however, appears to be largely unchanged as a result of entropic compensation for the decrease in enthalpy. This enthalpy-entropy compensation is attributed to changes in hydration of proteins in D2O compared to H2O. Analysis of thermodynamic data for the transfer of model compounds from H2O to D2O shows that almost all the changes in the enthalpy of unfolding and in the protein-ligand interactions due to water isotopic substitution can be rationalized by changes in hydration of the buried non-polar groups.

Calorimetry↗
PubMed1995

Three-dimensional structures of alpha and beta chemokines.

Members of the chemokine family of proteins play a key role in the orchestration of the immune response. This family has been further divided into two subfamilies, alpha and beta, based on sequence, function, and chromosomal location. To date, the three-dimensional structures of two members of the alpha subfamily, interleukin-8 (IL-8) and platelet factor 4, and one member of the beta subfamily, human macrophage inflammatory protein-1 beta (hMIP-1 beta), have been solved by either NMR or X-ray crystallography. In this review, we discuss their three-dimensional structures and their possible relationship to function. The structures of the monomers are very similar, as expected from the significant degree of sequence identity between these proteins. The quaternary structures of the alpha and beta chemokines, however, are entirely distinct and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1 beta dimer is elongated and cylindrical. Platelet factor 4 is a tetramer comprising a dimer of dimers of the IL-8 type. The IL-8 dimer comprises a six stranded anti-parallel beta-sheet, three strands contributed by each subunit, on top of which lie two antiparallel helices separated by approximately 14 A, and the symmetry axis is located between residues 26 and 26' (equivalent to residue 29 of hMIP-1 beta) at the center of strands beta 1 and beta 1'.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Structures of protein complexes by multidimensional heteronuclear magnetic resonance spectroscopy.

With the advent of multidimensional heteronuclear-edited and -filtered NMR experiments, the field of three-dimensional structure determination by NMR has again increased in scope, making it possible to move the technology beyond the approximately 10 kDa limit inherent to conventional two-dimensional NMR to systems up to potentially 35 to 40 kDa. This article outlines the basic strategies for solving three-dimensional structures of larger systems, in particular, protein complexes and multimeric proteins using three- and four-dimensional NMR spectroscopy, summarizes the key experiments, and illustrates the power of these methods using several examples of protein-DNA, protein-peptide complexes, and oligomeric proteins from the authors' laboratories.

Amino Acid Sequence↗
PubMed1994

A novel class of winged helix-turn-helix protein: the DNA-binding domain of Mu transposase.

BACKGROUND: Mu transposase (MuA) is a multidomain protein encoded by the bacteriophage Mu genome. It is responsible for translocation of the Mu genome, which is the largest and most efficient transposon known. While the various domains of MuA have been delineated by means of biochemical methods, no data have been obtained to date relating to its tertiary structure. RESULTS: We have solved the three-dimensional solution structure of the DNA-binding domain (residues 1-76; MuA76) of MuA by multidimensional heteronuclear NMR spectroscopy. The structure consists of a three-membered alpha-helical bundle buttressed by a three-stranded antiparallel beta-sheet. Helices H1 and H2 and the seven-residue turn connecting them comprise a helix-turn-helix (HTH) motif. In addition, there is a long nine-residue flexible loop or wing connecting strands B2 and B3 of the sheet. NMR studies of MuA76 complexed with a consensus DNA site from the internal activation region of the Mu genome indicate that the wing and the second helix of the HTH motif are significantly perturbed upon DNA binding. CONCLUSIONS: While the general appearance of the DNA-binding domain of MuA76 is similar to that of other winged HTH proteins, the connectivity of the secondary structure elements is permuted. Hence, the fold of MuA76 represents a novel class of winged HTH DNA-binding domain.

Bacteriophage mu↗
PubMed1994

Thermodynamics of unfolding of the all beta-sheet protein interleukin-1 beta.

The thermal denaturation of interleukin-1 beta in solution has been studied by differential scanning calorimetry at various pH values. It is shown that the thermal transition of interleukin-1 beta is completely reversible below pH 2.5, only partly reversible in the pH range 2.5-3.5, and irreversible above pH 3.5. Analysis of the reversible unfolding of interleukin-1 beta shows that the heat denaturation is well approximated by a two-state transition and is accompanied by a significant increase of heat capacity. The partial heat capacity of denatured interleukin-1 beta is very close to that expected for the completely unfolded protein. This permitted us to assign the thermodynamic characteristics of interleukin-1 beta denaturation to its complete unfolding and to correlate them with structural features of the protein. The contributions of hydrogen bonding and hydrophobic interactions to the stability of interleukin-1 beta are analyzed and compared to those for other globular proteins. It is shown that the Gibbs energy of a hydrogen bond in a beta-sheet structure is greater than in alpha-helices.

Calorimetry, Differential Scanning↗
PubMed1994

High-resolution structure of Ascaris trypsin inhibitor in solution: direct evidence for a pH-induced conformational transition in the reactive site.

BACKGROUND: The Ascaris trypsin inhibitor (ATI) is a member of a new family of serine protease inhibitors isolated from the helminthic worm Ascaris lumbricoides var suum. This family comprises five chymotrypsin/elastase inhibitors and one trypsin inhibitor. Members are characterized by the presence of five disulfide bonds (two of which are located on either side of the reactive site) in a single small protein domain of 61-62 residues. RESULTS: The solution structure of ATI has been determined at pH 2.4 and pH 4.75 by NMR spectroscopy. Iterative refinement permitted the unambiguous assignment of the pairing of the five disulfide bridges (Cys5-Cys38, Cys15-Cys33, Cys18-Cys29, Cys22-Cys60, and Cys40-Cys54) which were previously unknown. The structure includes four short beta-strands arranged in two approximately perpendicular beta-sheets. The reactive site loop is bounded by two disulfide bridges (Cys15-Cys33 and Cys18-Cys29) and is part of the long loop (residues 15-25) connecting strands beta 1 and beta 2. Comparison of the nuclear Overhauser enhancement data at the two pH values revealed significant differences centered around the reactive site. While the reactive site at pH 2.4 closely resembles that of other protease inhibitors, at pH 4.75 the reactive site loop undergoes a major conformational rearrangement involving the psi backbone torsion angles of the P2, P1 and P1' residues (residues 30-32). This is associated with a change in the positions of the side chains of Arg31 and Glu32. CONCLUSIONS: The overall three-dimensional structure of ATI posesses an unusual fold and, with the exception of the reactive site, shows no similarity to other serine protease inhibitors. The observation that the reactive site of the low pH form of ATI is similar to that of other serine proteases suggests that this is the active form of the protein.

Animals↗
PubMed1994

High-resolution structure of the oligomerization domain of p53 by multidimensional NMR.

The three-dimensional structure of the oligomerization domain (residues 319 to 360) of the tumor suppressor p53 has been solved by multidimensional heteronuclear magnetic resonance (NMR) spectroscopy. The domain forms a 20-kilodalton symmetric tetramer with a topology made up from a dimer of dimers. The two primary dimers each comprise two antiparallel helices linked by an antiparallel beta sheet. One beta strand and one helix are contributed from each monomer. The interface between the two dimers forming the tetramer is mediated solely by helix-helix contacts. The overall result is a symmetric, four-helix bundle with adjacent helices oriented antiparallel to each other and with the two antiparallel beta sheets located on opposing faces of the molecule. The tetramer is stabilized not only by hydrophobic interactions within the protein core but also by a number of electrostatic interactions. The implications of the structure of the tetramer for the biological function of p53 are discussed.

Base Sequence↗
PubMed1994

The high-resolution three-dimensional solution structures of the oxidized and reduced states of human thioredoxin.

BACKGROUND: Thioredoxin is a ubiquitous protein and is involved in a variety of fundamental biological functions. Its active site is conserved and has two redox active cysteines in the sequence Trp-Cys-Gly-Pro-Cys. No structures of the oxidized and reduced states from the same species have been determined at high resolution under the same conditions and using the same methods. Hence, any detailed comparison of the two oxidation states has been previously precluded. RESULTS: The reduced and oxidized states of the (C62A, C69A, C73A) mutant of human thioredoxin have been investigated by multidimensional heteronuclear NMR. Structures for both states were determined on the basis of approximately 28 experimental restraints per residue, and the resulting precision of the two structures is very high. Consequently, subtle differences between the oxidized and reduced states can be reliably assessed and evaluated. Small differences, particularly within and around the active site can be discerned. CONCLUSIONS: Overall, the structures of the reduced and oxidized states of the (C62A, C69A, C73A) mutant of human thioredoxin are very similar (with a backbone atomic root mean square difference of about 0.9 A) and the packing of side chains within the protein core is nearly identical. The conformational change between oxidized and reduced human thioredoxin is very small and localized to areas in spatial proximity to the redox active cysteines. These subtle structural differences, in addition to the restriction of conformational freedom within the active site upon oxidation, may be important for the different activities of thioredoxin involving a variety of target proteins.

Amino Acid Sequence↗
PubMed1994

Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins.

BACKGROUND: Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction. RESULTS: We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences. CONCLUSIONS: Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.

Bacterial Proteins↗
PubMed1994

Refolding proteins by gel filtration chromatography.

We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography. We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS-1 protein, bovine ribonuclease A and E. coli integration host factor can be refolded into the native conformation using this technique. We have extended this strategy to the preparation of milligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X-ray crystallography. The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.

Animals↗
PubMed1994

High-resolution solution structure of the beta chemokine hMIP-1 beta by multidimensional NMR.

The three-dimensional structure of a member of the beta subfamily of chemokines, human macrophage inflammatory protein-1 beta (hMIP-1 beta), has been determined with the use of solution multidimensional heteronuclear magnetic resonance spectroscopy. Human MIP-1 beta is a symmetric homodimer with a relative molecular mass of approximately 16 kilodaltons. The structure of the hMIP-1 beta monomer is similar to that of the related alpha chemokine interleukin-8 (IL-8). However, the quaternary structures of the two proteins are entirely distinct, and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1 beta dimer is elongated and cylindrical. This provides a rational explanation for the absence of cross-binding and reactivity between the alpha and beta chemokine subfamilies. Calculation of the solvation free energies of dimerization suggests that the formation and stabilization of the two different types of dimers arise from the burial of hydrophobic residues.

Amino Acid Sequence↗
PubMed1994

Structural comparisons among the short-chain helical cytokines.

BACKGROUND: Cytokines and growth factors are soluble proteins that regulate the development and activities of many cell types. One group of these proteins have structures based on a four-helix bundle, though this similarity is not apparent from amino acid sequence comparisons. An understanding of how diverse sequences can adopt the same fold would be useful for recognizing and aligning distant homologs and for applying structural information gained from one protein to other sequences. RESULTS: We have approached this problem by comparing the five known structures which adopt a granulocyte-macrophage colony-stimulating factor (GM-CSF)-like, or short-chain fold: interleukin (IL)-4, GM-CSF, IL-2, IL-5, and macrophage colony-stimulating factor. The comparison reveals a common structural framework of five segments including 31 inner-core and 30 largely exposed residues. Buried polar interactions found in each protein illustrate how complementary substitutions maintain protein stability and may help specify unique core packing. A profile based on the known structures is not sufficient to guarantee accurate amino acid sequence alignments with other family members. Comparisons of the conserved short-chain framework with growth hormone define the optimal structural alignment. CONCLUSIONS: Our results are useful for extrapolating functional results among the short-chain cytokines and growth hormone, and provide a foundation for similar characterization of other subfamilies. These results also show that the placement of polar residues at different buried positions in each protein complicates sequence comparisons, and they document a challenging test case for methods aimed at recognizing and aligning distant homologs.

Amino Acid Sequence↗
PubMed1994

Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure and folding topology of the first RNA binding domain of the human hnRNP A1 protein was determined by multidimensional heteronuclear NMR spectroscopy. The 92 amino acid long domain exhibits a beta alpha beta beta alpha beta folding pattern, arranged in a four-stranded antiparallel beta-sheet flanked by two alpha-helices, which is very similar to that found for other members of this family. Regions of marked variation between the structurally characterized RNA binding proteins of this class to date are mainly localized in the loops connecting the secondary structure elements.

Amino Acid Sequence↗
PubMed1994

Localization of bound water in the solution structure of a complex of the erythroid transcription factor GATA-1 with DNA.

BACKGROUND: The erythroid specific transcription factor GATA-1 is responsible for the regulation of transcription of erythroid-expressed genes and is an essential component required for the generation of the erythroid lineage. GATA-1 binds specifically as a monomer to the asymmetric consensus target sequence (T/A)GATA-(A/G) found in the cis-regulatory elements of all globin genes and most other erythroid specific genes that have been examined. We have previously determined the solution structure of the complex of the zinc-containing DNA-binding domain of chicken GATA-1 with its cognate DNA target site by multidimensional heteronuclear NMR. From previous studies of complexes between proteins and DNA, water appears to play an important role in DNA-protein recognition by mediating bridging hydrogen bonds between functional groups on the protein and DNA bases. Solvation free energy calculations, however, suggest that hydrophobic interactions should exclude water from parts of the GATA-1:DNA interface. RESULTS: Using water-selective two-dimensional heteronuclear magnetic resonance spectroscopy, we have identified the location of bound water molecules in the specific complex of chicken GATA-1 with DNA. A number of water molecules could be detected between the protein and the phosphate backbone, as well as at the solvent exposed surface of the protein. However, no water molecules could be observed at the interface of the protein with the bases of the DNA. With only one exception, the bound water molecules have a residency time > 200-300 ps. CONCLUSIONS: Unlike other protein-DNA complexes, the majority of specific interactions between GATA-1 and the DNA bases in the major groove are hydrophobic in nature. The exclusion of water from the protein-DNA base interface in the major groove supports the view that the specific binding energy is indeed dominated by hydrophobic effects.

Animals↗
PubMed1994

Mapping the binding surface of interleukin-8 complexed with an N-terminal fragment of the type 1 human interleukin-8 receptor.

Interleukin-8 and its receptors are key mediators of immune and inflammatory responses. Heteronuclear NMR spectroscopy has been utilized to map the binding surface on interleukin-8 (IL-8) for an N-terminal fragment of the human Type-1 IL-8 receptor. A peptide corresponding to residues 1-40 of the IL-8 type 1 receptor (IL8-r1) was titrated into a sample of uniformly 15N-labeled IL-8. IL8-r1 binds to IL-8 with a dissociation constant of 170 +/- 50 microM assuming the peptide binds with a stoichiometry of one peptide per IL-8 monomer, exchanges rapidly (> 900 s-1) between free and bound states, and selectively perturbs the chemical environment of several IL-8 residues. The binding surface on IL-8 suggested by our results is comprised of residues in strand beta 3 of the beta-sheet (Glu48 to Cys50), the turn preceding beta 3 (Ser44), the C-terminal alpha-helix (Val61) and the irregular N-terminal loop region (Thr12, Lys15, Phe17, His18, Lys20 and Phe21). The IL-8 dimer appears to present two symmetrical binding surfaces for the IL8-r1 peptide, suggesting two receptor peptides may bind per dimer.

Amino Acid Sequence↗
PubMed1994

Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy.

The backbone dynamics of the immunoglobulin-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an analysis of the T1/T2 ratios was 3.3 ns at 26 degrees C. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheet arranged in a -1, +3x, -1 topology, on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A number of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (< 20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40 degrees) jumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1994

Young Investigator Award Lecture. Structures of larger proteins, protein-ligand and protein-DNA complexes by multidimensional heteronuclear NMR.

The recent development of a whole panoply of multidimensional heteronuclear-edited and -filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the 10-kDa limit of conventional 2-dimensional NMR to systems up to potentially 35-40 kDa. The basic strategy for solving 3-dimensional structures of larger proteins and protein-ligand complexes in solution using 3- and 4-dimensional NMR spectroscopy is summarized, and the power of these methods is illustrated using 3 examples: interleukin-1 beta, the complex of calmodulin with a target peptide, and the specific complex of the transcription factor GATA-1 with its cognate DNA target site.

Amino Acid Sequence↗