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G M Clore

Publications and source records attributed to G M Clore.

At least 55 records · Page 3Linked to original sources
PubMed1998

Determination of three-bond 1H3'-31P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy.

A new sensitive two-dimensional quantitative J correlation experiment is described for measuring 3JH3'-P couplings in nucleic acids and protein-nucleic acid complexes. The method is based on measuring the change in intensity of the 1H-1H cross peaks in a constant-time 1H-1H COSY experiment which occurs in the presence and absence of 3JH3'-P dephasing during the constant-time evolution period. For protein-nucleic acid complexes where the protein is 13C-labeled but the nucleic acid is not, 12C-filtering is readily achieved by the application of a series of 13C purge pulses during the constant time evolution period without any loss of signal-to-noise of the nucleic acid cross peaks. The method is demonstrated for the Dickerson DNA dodecamer and a 19 kDa complex of the transcription factor SRY with a 14mer DNA duplex. The same approach should be equally applicable to numerous other problems, including the measurement of JH-Cd couplings in cadmium-ligated proteins, or 3JCH couplings in other selectively enriched compounds.

Base Sequence↗
PubMed1998

3D NMR experiments for measuring 15N relaxation data of large proteins: application to the 44 kDa ectodomain of SIV gp41.

A suite of 3D NMR experiments for measuring 15N-¿1H¿ NOE, 15N T1, and 15N T1rho values in large proteins, uniformly labeled with 15N and 13C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D 1H-15N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated 13C/15N-labeled sample of the 44 kDa ectodomain of SIV gp41.

Carbon Isotopes↗
PubMed1998

Determining the structures of large proteins and protein complexes by NMR.

Recent advances in multidimensional NMR methodology to obtain 1H, 15N and 13C resonance assignments, interproton-distance and torsion-angle restraints, and restraints that characterize long-range order have, coupled with new methods of structure refinement, permitted solution structure of proteins in excess of 250 residues to be solved. These developments may permit the determination by NMR of the structures of macromolecules up to 50-60kDa, thereby bringing into reach numerous systems of considerable biological interest, including a large variety of protein-protein and protein-nucleic-acid complexes.

Magnetic Resonance Spectroscopy↗
PubMed1998

NMR structure determination of proteins and protein complexes larger than 20 kDa.

Recent advances in multidimensional nuclear magnetic resonance methodology to obtain 1H, 15N and 13C resonance assignments, interproton distance and torsion angle restraints, and restraints that characterize long-range order, coupled with new methods of structure refinement and novel methods for reducing linewidths, have permitted three-dimensional solution structures of single chain proteins in excess of 250 residues and multimeric protein in excess of 40 kDa to be solved. These developments may permit the determination by nuclear magnetic resonance of macromolecular structures up to molecular weights in the 50-60 kDa range, thereby bringing into reach numerous systems of considerable biological interest, including a large variety of protein-protein and protein-nucleic acid complexes.

Carbon Isotopes↗
PubMed1998

An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli.

A cost-effective protocol for uniform 15N and/or 13C isotope labeling of bacterially expressed proteins is presented. Unlike most standard protocols, cells are initially grown in a medium containing nutrients at natural abundance and isotopically labeled nutrients are only supplied at the later stages of growth and during protein expression. This permits the accumulation of a large cell mass without the need to employ expensive isotopically labeled nutrients. The abrupt decrease in oxygen consumption that occurs upon complete exhaustion of essential nutrients is used to precisely time the switch between unlabeled and labeled nutrients. Application of the protocol is demonstrated for wild-type and a mutant of the N-terminal zinc-binding domain of HIV-1 integrase.

Carbon Isotopes↗
PubMed1998

Solution structure of the cellular factor BAF responsible for protecting retroviral DNA from autointegration.

The solution structure of the human barrier-to-autointegration factor, BAF, a 21,000 Mr dimer, has been solved by NMR, including extensive use of dipolar couplings which provide a priori long range structural information. BAF is a highly evolutionarily conserved DNA binding protein that is responsible for inhibiting autointegration of retroviral DNA, thereby promoting integration of retroviral DNA into the host chromosome. BAF is largely helical, and each subunit is composed of five helices. The dimer is elongated in shape and the dimer interface comprises principally hydrophobic contacts supplemented by a single salt bridge. Despite the absence of any sequence similarity to any other known protein family, the topology of helices 3-5 is similar to that of a number of DNA binding proteins, with helices 4 and 5 constituting a helix-turn-helix motif. A model for the interaction of BAF with DNA that is consistent with structural and mutagenesis data is proposed.

Amino Acid Sequence↗
PubMed1998

Solution structure of cyanovirin-N, a potent HIV-inactivating protein.

The solution structure of cyanovirin-N, a potent 11,000 Mr HIV-inactivating protein that binds with high affinity and specificity to the HIV surface envelope protein gp120, has been solved by nuclear magnetic resonance spectroscopy, including extensive use of dipolar couplings which provide a priori long range structural information. Cyanovirin-N is an elongated, largely beta-sheet protein that displays internal two-fold pseudosymmetry. The two sequence repeats (residues 1-50 and 51-101) share 32% sequence identity and superimpose with a backbone atomic root-mean-square difference of 1.3 A. The two repeats, however, do not form separate domains since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Analysis of surface hydrophobic clusters suggests the location of potential binding sites for protein-protein interactions.

Amino Acid Sequence↗
PubMed1998

Minor groove-binding architectural proteins: structure, function, and DNA recognition.

To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box-binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box-containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein-DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.

Animals↗
PubMed1997

Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains.

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end-binding domain (Ibetagamma). The three-dimensional solution structure of the Ibeta subdomain (residues 77-174) has been determined using multidimensional NMR spectroscopy. It comprises five alpha-helices, including a helix-turn-helix (HTH) DNA-binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc-like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2-4 is very similar to that of helices 1-3 of the previously determined solution structure of the MuA Igamma subdomain and to that of the homeodomain family of HTH DNA-binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Ibeta to the more conserved Mu end distal half (beta subsite) and Igamma to the Mu end proximal half (gamma subsite) of the consensus Mu end-binding site. The complete Ibetagamma domain binds the recognition sequence with a 100- to 1000-fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA-binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Ibeta and Igamma subdomains, a model for the interaction of the complete Ibetagamma domain with DNA is proposed.

Amino Acid Sequence↗
PubMed1997

Solution structure of the I gamma subdomain of the Mu end DNA-binding domain of phage Mu transposase.

The MuA transposase of phase Mu is a large modular protein that plays a central role in transposition. We show that the Mu end DNA-binding domain, I beta gamma, which is responsible for binding the DNA attachment sites at each end of the Mu genome, comprises two subdomains, I beta and I gamma, that are structurally autonomous and do not interact with each other in the absence of DNA. The solution structure of the I gamma subdomain has been determined by multidimensional NMR spectroscopy. The structure of I gamma comprises a four helix bundle and, despite the absence of any significant sequence identity, the topology of the first three helices is very similar to that of the homeodomain family of helix-turn-helix DNA-binding proteins. The helix-turn-helix motif of I gamma, however, differs from that of the homeodomains in so far as the loop is longer and the second helix is shorter, reminiscent of that in the POU-specific domain.

Amino Acid Sequence↗
PubMed1997

Determination of the secondary structure and global topology of the 44 kDa ectodomain of gp41 of the simian immunodeficiency virus by multidimensional nuclear magnetic resonance spectroscopy.

The gp41 protein of the human (HIV) and simian (SIV) immunodeficiency viruses is part of the envelope glycoprotein complex gp41/gp120 which plays an essential role in viral infection. We present a multidimensional NMR study on the trimeric 44 kDa soluble ectodomain of SIV gp41 (e-gp41), comprising residues 27 to 149. Despite the large molecular weight and very limited spectral dispersion, complete backbone 1H, 13C, 13CO and 15N assignments have been made using a combination of triple resonance experiments on uniformly 13C/15N and 2H/13C/15N-labeled samples. The secondary structure of SIV e-gp41, derived on the basis of 13C chemical shifts, NH exchange rates, medium range nuclear Overhauser enhancements (NOE), and 3JHNalpha coupling constants, consists of a 49 residue helix at the N terminus (residues 29 to 77) and a 40 residue helix at the C terminus (residues 108 to 147), connected by a 30 residue loop which does not display any of the characteristics of regular secondary structure. The cross-peak intensities of the loop region in scalar correlation experiments suggests that it is more mobile than the core helical regions. The presence, however, of numerous long range NOEs, both intra and inter-subunit, within the loop indicates that it adopts a well-defined structure in which the loops from the three subunits interact with each other. Based on a number of long range intra and inter-subunit NOEs, a topological model is presented for the symmetric SIV e-gp41 trimer in which the N-terminal helices are packed within the protein interior in a parallel trimeric coiled-coil arrangement, while the C-terminal helices are located on the protein exterior, oriented antiparallel to the N-terminal helices.

Amino Acid Sequence↗
PubMed1997

Architectural accommodation in the complex of four p53 DNA binding domain peptides with the p21/waf1/cip1 DNA response element.

High resolution chemical footprinting and cross-linking experiments have provided a basis for elucidating the overall architecture of the complex between the core DNA binding domain of p53 (p53DBD, amino acids 98-309) and the p21/waf1/cip1 DNA response element implicated in the G1/S phase cell cycle checkpoint. These studies complement both a crystal structure and earlier biophysical studies and provide the first direct experimental evidence that four subunits of p53DBD bind to the response element in a regular staggered array having pseudodyad symmetry. The invariant guanosines in the highly conserved C(A/T)|(T/A)G parts of the consensus half-sites are critical to the p53DBD-DNA binding. Molecular modeling of the complex using the observed peptide-DNA contacts shows that when four subunits of p53DBD bind the response element, the DNA has to bend approximately 50 degrees to relieve steric clashes among different subunits, consistent with recent DNA cyclization studies. The overall lateral arrangement of the four p53 subunits with respect to the DNA loop comprises a novel nucleoprotein assembly that has not been reported previously in other complexes. We suggest that this kind of nucleoprotein superstructure may be important for p53 binding to response elements packed in chromatin and for subsequent transactivation of p53-mediated genes.

Base Sequence↗
PubMed1997

The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.

Amino Acid Sequence↗
PubMed1997

Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system.

The interaction between the approximately 30 kDa N-terminal domain of enzyme I (EIN) and the approximately 9.5 kDa histidine-containing phosphocarrier protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been investigated by heteronuclear magnetic resonance spectroscopy. The complex is in fast exchange, permitting us to follow the chemical shift changes of the backbone NH and 15N resonances of EIN upon complex formation by recording a series of 1H-15N correlation spectra of uniformly 15N-labeled EIN in the presence of increasing amounts of HPr at natural isotopic abundance. The equilibrium association constant derived from analysis of the titration data is approximately 1.5 x 10(5) M(-1), and the lower limit for the dissociation rate constant is 1100 s(-1). By mapping the backbone chemical shift perturbations on the three-dimensional solution structure of EIN [Garrett, D. S., Seok, Y.-J., Liao, D.-I., Peterkofsky, A., Gronenborn, A. M., & Clore, G. M. (1997) Biochemistry 36, 2517-2530], we have identified the binding surface of EIN in contact with HPr. This surface is primarily located in the alpha domain and involves helices H1, H2, and H4, as well as the hinge region connecting helices H2 and H2'. The data also indicate that the active site His 15 of HPr must approach the active site His 189 of EIN along the shallow depression at the interface of the alpha and alpha/beta domains. Interestingly, both the backbone and side chain resonances (assigned from a long-range 1H-15N correlation spectrum) of His 189, which is located at the N-terminus of helix H6 in he alpha/beta domain, are only minimally perturbed upon complexation, indicating that His 189 (in the absence of phosphorylation) does not undergo any significant conformational change or change in pK(a) value upon HPr binding. On the basis of results of this study, as well as a previous study which delineated the interaction surface for EI on HPr [van Nuland, N. A. J., Boelens, R., Scheek, R. M., & Robillard, G. T. (1995) J. Mol. Biol. 246, 180-193], a model for the EIN/HPr complex is proposed in which helix 1 (residues 16-27) and the helical loop (residues 49-53) of HPr slip between the two pairs of helices constituting the alpha domain of EIN. In addition, we suggest a functional role for the kink between helices H2 and H2' of EIN, providing a flexible joint for this interaction to take place.

Bacterial Proteins↗
PubMed1997

Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR.

The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy. Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II. To facilitate and confirm NH, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths. Ninety-eight percent of the 1H, 15N, and 13C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments. The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms. The structure is ellipsoidal in shape, approximately 78 A long and 32 A wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha-domain (residues 33-143) consisting of four helices. The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N- and C-terminal domains of intact enzyme I. A comparison with the recently solved X-ray structure of EIN [Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B. R., Peterkofsky, A., & Davies, D. R. (1996) Structure 4, 861-872] indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates. The active site His189 is located in a cleft at the junction of the alpha and alpha/beta domains and has a pKa of approximately 6.3. His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation.

Amino Acid Sequence↗
PubMed1997

The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.

Chromatography, Gel↗