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G F King

Publications and source records attributed to G F King.

At least 37 records · Page 2Linked to original sources
PubMed1997

Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Bacillus subtilis↗
PubMed1997

The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Amino Acid Sequence↗
PubMed1996

1H-NMR visible neutral lipids in activated T lymphocytes: relationship to phosphatidylcholine cycling.

Two-dimensional 1H-NMR spectroscopy was used to compare changes in the concentration of isotropically-tumbling neutral lipid during the activation of splenic and thymic T lymphocytes. The concentration of mobile neutral lipid (MNL) was similar in splenic and thymic T cells after 72 h of activation with phorbol myristate acetate and ionomycin. However, after 120 h of activation, MNL concentrations in splenic T cells were more than 3-fold higher than in thymic T cells. An increase in choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) was also observed in both thymic and splenic T cells after 24 h of activation. However, after 72 h of stimulation, Cho and PCho levels had decreased and continued to decline at 96-120 h, while GPC continued to be maintained at elevated levels. The simultaneous increase in MNL and GPC and the decline in Cho and PCho leads us to propose that the synthesis of NMR-visible MNL in activated lymphocytes is linked to the phosphatidylcholine cycle.

Animals↗
PubMed1996

Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins.

c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36 --> E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., & Easterbrook-Smith, S.B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 microM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.

Amino Acid Sequence↗
PubMed1996

The Bacillus subtilis DNA replication terminator.

The recent discovery of the Bacillus subtilis plasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B. subtilis replication terminators in general. The minimal TerI and TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators. It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region. A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site. The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator. TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator. It was found that the deviations introduced converted the terminator significantly towards polarity of action. The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence. The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB. A model to account for the generation of RTP-terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI-VI is presented.

Bacillus subtilis↗
PubMed1996

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer.

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel alpha-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.

Amino Acid Sequence↗
PubMed1996

Backbone dynamics of the c-Jun leucine zipper: 15N NMR relaxation studies.

The backbone dynamics of the coiled-coil leucine zipper domain of c-Jun have been studied using proton-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation times, together with {1H}15N NOEs, were measured and analyzed by considering the protein to approximate a prolate ellipsoid. An analysis of the T1/T2 ratios for residues in the well-structured section of the protein showed that a model for the spectral density function in which the protein is considered to reorient anisotropically fitted the data significantly better than an isotropic model. Order parameters (S2) in the range 0.7-0.9 were observed for most residues, with lower values near the C-terminus, consistent with fraying of the two helices comprising the coiled-coil. Because nearly all of the N-H vectors have small angles to the long axis of the molecule, there was some uncertainty in the value of the rotational diffusion coefficient Dpar, which describes rotation about the long axis. Thus, an alternative method was examined for its ability to provide independent estimates of Dpar and Dperp (the diffusion coefficient describing rotation about axes perpendicular to the long axis); the transitional diffusion coefficient (Dt) of the protein was measured, and hydrodynamic calculations were used to predict Dpar and Dperp. However, the derived rotational diffusion coefficients proved to be very dependent on the hydrodynamic model used to relate Dt to Dpar and Dperp, and consequently the values obtained from the T1/T2 analysis were used in the order-paramenter analysis. Although it has previously been reported that the side chain of a polar residue at the dimer interface, Asn22, undergoes a conformational exchange process and destabilizes the dimer, no evidence of increased backbone mobility in this region was detected, suggesting that this process is confined to the Asn side chain.

Amino Acid Sequence↗
PubMed1995

Nuclear magnetic resonance characterization of the Jun leucine zipper domain: unusual properties of coiled-coil interfacial polar residues.

Leucine zippers constitute a widely observed structural motif which serves to promote both homo- and heterodimerization in a number of DNA-binding proteins. As part of our ongoing efforts to characterize both the structure and the dynamical properties of this dimerization domain as they relate to biological function, we report here the secondary structure in solution of a recombinant dimeric peptide (rJunLZ) comprising residues Arg276-Asn314 of the leucine zipper domain of c-Jun. Two- and three-dimensional homo- and heteronuclear NMR experiments have allowed definition of the secondary structure of rJunLZ and have provided a total of approximately 1500 interproton distance and 62 phi dihedral angle constraints for tertiary structure calculations. Amide proton protection factors, calculated from hydrogen-deuterium exchange experiments, have identified 62 hydrogen bonds in the rJunLZ dimer. We have also examined the role of Asn22, the only polar residue situated at the hydrophobic dimer interface. Virtually all leucine zipper sequences contain such a polar residue (usually Asn) near the center of the motif. X-ray crystallographic studies showed that, in the case of the GCN4 homodimer, the polar residue (Asn) adopts an asymmetric conformation in an otherwise essentially symmetric structure. In contrast, all NMR studies of leucine zipper homodimers to date have suggested that the dimers are completely symmetric in solution. We present evidence that the side-chain amide protons of Asn22 are hydrogen-bonded in solution and that this side chain exchanges rapidly between two distinct conformations. On the basis of these observations, we propose a dynamic model which can explain the apparent differences in symmetry observed in NMR and X-ray crystallographic studies of leucine zipper homodimers. We show that mutation of Asn22 to a hydrophobic Leu residue markedly increases the thermal stability of the rJunLZ homodimer, consistent with a destabilizing role for this residue. However, at temperatures below 30 degrees C, the Asn22-->Leu mutant rearranges to form oligomers larger than the dimer, as was previously observed for the corresponding Asn-->Val mutation in the GCN4 leucine zipper. These results are consistent with the hypothesis that the polar Asn residue commonly observed at the interface of leucine zippers imposes specificity for the dimer structure at the expense of stability [Harbury, P.B., Zhang, T., Kim, P.S., & Alber, T. (1993) Science 262, 1401-1407].

Amino Acid Sequence↗
PubMed1995

Structural and biochemical studies of human galanin: NMR evidence for nascent helical structures in aqueous solution.

The 30-residue human neuropeptide, galanin, was shown to bind to rat insulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insulin secretion from these cells in a manner quantitatively similar to that of porcine galanin. Neither human nor porcine galanin stimulated Ca2+ mobilization in cultured human small cell lung carcinoma cells. Sedimentation equilibrium analysis of human galanin showed that it was strictly monomeric in aqueous solution, indicating that the peptide interacts with its receptor(s) as a monomer. The monomeric nature of the peptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on galanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equilibrium with random coil. At 33 degrees C, the short-range structures include a nascent helix spanning residues 3-11 which incorporates a hydrophobic core from residues 6-11. Residues 14-18 and 22-30 display sequential NH-NH and C beta H-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the alpha-region of conformational space. However, no medium-range dipolar connectivities indicative of nascent helix or turn conformations were observed. At 3 degrees C, almost all residues significantly populate the alpha-region of conformational space, and the nascent helix between residues 3 and 11, with its hydrophobic core, is retained.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: application to myosin light chain 2.

At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of approximately 7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 corresponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D 15N-1H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T2 value for MLC2 increased from approximately 30 ms in the absence of CHAPS to approximately 56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules.

Animals↗
PubMed1994

The solution structure of sarafotoxin-c. Implications for ligand recognition by endothelin receptors.

The solution structure of sarafotoxin-c has been determined using NMR spectroscopy. A total of 112 interproton distance constraints derived from two-dimensional NMR spectra were used to calculate a family of structures using a combination of distance geometry and dynamical simulated annealing calculations. The structures reveal a well defined alpha-helix extending from Glu9 to Cys15 and an N-terminal region (Cys1-Asp8) that is tightly constrained by disulfide bonds to Cys residues in the central helix. In contrast, the C-terminal region (His16-Trp21) does not adopt a defined conformation in the final family of structures. This is consistent with the paucity of NMR-derived structural constraints obtained for this region and leads to the suggestion that the C-terminal region oscillates rapidly between a number of substantially different conformers. It is proposed that differences between the central helix of the endothelin and sarafotoxin isopeptides might be important in binding of these ligands by the G protein-coupled endothelin receptors.

Amino Acid Sequence↗
PubMed1994

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain.

Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (approximately 15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel alpha-helices. We also present 15N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.

Amino Acid Sequence↗
PubMed1994

Theoretical and practical aspects of NMR studies of cells.

Various theoretical and practical aspects of biological nuclear magnetic resonance (NMR) spectroscopy that are relevant to the study of immune cells are discussed as a prelude to the following papers in this issue of ImmunoMethods. We explain some of the salient features of modern Fourier-transform NMR spectroscopy, including spectral acquisition, Fourier transformation, signal averaging, apodization, and relaxation phenomena. The major features of the one-dimensional NMR spectrum are summarized prior to a brief description of two-dimensional NMR spectroscopy. We consider various practical questions, such as which NMR-receptive nuclide might be most useful for discerning the metabolic information being sought; in particular, the relative advantages and disadvantages of 1H, 13C, 19F, and 31P NMR spectroscopy are discussed in the context of elucidating metabolic information. The relative merits and pitfalls of using cell extracts, cell suspensions, and perfused immobilized cells for studies of immune cell activation are also considered.

Animals↗
PubMed1994

Two-dimensional 1H NMR studies of membrane changes during the activation of primary T lymphocytes.

Two-dimensional 1H NMR spectroscopy was used to quantify the level of "mobile" plasma membrane triglyceride and the intracellular concentrations of water-soluble phospholipid precursors during the activation of both mature and immature primary T lymphocytes. The concentration of mobile triglyceride in the plasma membrane was seen to increase approximately 35-fold during 72 h of activation of murine thymic and splenic T lymphocytes with ionomycin and phorbol 12-myristate 13-acetate. This dramatic increase in mobile plasma membrane triglyceride during the activation of both mature and immature T-lymphocyte populations supports the hypothesis that immune cell activation is associated with increased plasma membrane fluidity. The intracellular concentrations of various phospholipid precursors were shown to increase during the early stages of T-lymphocyte activation and then remain at levels above those in resting cells. This may facilitate de novo phospholipid biosynthesis, which is presumably necessary since cell volume, and hence the plasma membrane surface area, was demonstrated to increase significantly during T-lymphocyte activation. Various models that might explain the origin of the NMR-visible plasma membrane triglyceride that is observed during immune cell activation and malignant transformation are examined.

Animals↗
PubMed1993

Symmetry and secondary structure of the replication terminator protein of Bacillus subtilis: sedimentation equilibrium and circular dichroic, infrared, and NMR spectroscopic studies.

We have used analytical ultracentrifugation in combination with a number of spectroscopic techniques to analyze the symmetry and secondary structure of the DNA-binding replication terminator protein (RTP) of Bacillus subtilis. Sedimentation equilibrium studies confirm that RTP is a dimer in solution under the conditions used for spectroscopic analysis, whereas the number of cross peaks displayed in 1H-15N HSQC NMR spectra of uniformly 15N-labeled RTP are consistent with the primary structure of the monomer. These two results in combination lead to the conclusion that RTP is a symmetric dimer in solution. Circular dichroic and Fourier-transform infrared spectra reveal, in contrast to the results obtained from a number of commonly used secondary structure prediction algorithms, that RTP contains 20-30% alpha-helical and 40-50% beta-sheet/beta-turn secondary structure and that the conformation of the protein remains unchanged over the pH range 5-8. It is proposed on the basis of protein folding-class prediction algorithms, in combination with various physical properties of RTP, that it belongs to the alpha + beta protein-folding class.

Algorithms↗
PubMed1993

The solution structure of the leucine zipper motif of the Jun oncoprotein homodimer.

Proton NMR studies have been performed on a 9.8-kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun to ascertain its conformation in aqueous solution. Analysis of two-dimensional scalar and dipolar-coupling experiments enabled almost all proton resonances to be sequence-specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric dimer in solution, consistent with the formation of a coiled-coil arrangement of parallel alpha-helical strands. The rates of exchange of individual amide protons with solvent, as well as hydrogen-bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled-coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled-coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain.

Amino Acid Sequence↗