Matthew Revington

A pulsed field gradient isotope‐filtered 3D 13C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

A pulsed field gradient three‐dimensional isotope‐filtered 13C HMQC‐NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp‐repressor, its natural abundance co‐repressor, l‐tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra‐ and intermolecular NOEs in the isotope‐edited 3D 13C HMQC‐NOESY spectrum of the complex.

The second stalk of Escherichia coli ATP synthase.

Two stalks link the F(1) and F(0) sectors of ATP synthase. The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex. The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx. 125 A from the membrane. This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis. In vitro, b(2)delta is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with delta. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair. However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.

The Dimerization Domain of the b Subunit of theEscherichia coli F1F0-ATPase

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F1F0 ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53–122 sequence. Sedimentation velocity and 15N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103–110 failed to form disulfides either with the identical mutant or when mixed with the other 103–110 cysteine mutants. These studies establish that the bsubunit dimer depends on interactions that occur between residues in the 53–122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

The b subunit of Escherichia coli ATP synthase.

The b subunit of ATP synthase is a major component of the second stalk connecting the F1and F0 sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF1, where they interact with the δ subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal δ-binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the δ-binding domain is more globular. Sites ofcrosslinking between b and the a, α, β, and δ subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.

SAGA: rapid automatic mainchain NMR assignment for large proteins

Here we describe a new algorithm for automatically determining the mainchain sequential assignment of NMR spectra for proteins. Using only the customary triple resonance experiments, assignments can be quickly found for not only small proteins having rather complete data, but also for large proteins, even when only half the residues can be assigned. The result of the calculation is not the single best assignment according to some criterion, but rather a large number of satisfactory assignments that are summarized in such a way as to help the user identify portions of the sequence that are assigned with confidence, vs. other portions where the assignment has some correlated alternatives. Thus very imperfect initial data can be used to suggest future experiments.

Folding and stability of the b subunit of the F1F0 ATP synthase

The F1F0 ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F0) and soluble (F1) sectors. The three‐dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F1F0 complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N‐ and C‐terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two‐state, cooperative unfolding with Tm values between 30 and 40°C. CD spectra for the region comprising residues 53–122 (b53–122) showed θ222/θ208 = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b53–122 (ΔG, ΔH and ΔCp) were similar to those reported for several nonideal, coiled‐coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled‐coil at the interface of the b dimer.

Analysis of the structure of human apo-S100B at low temperature indicates a unimodal conformational distribution is adopted by calcium-free S100 proteins.

S100B is one of the best‐characterized members of the calcium‐signaling S100 protein family. Most S100 proteins are dimeric, with each monomer containing two EF‐hand calcium‐binding sites (EF1, EF2). S100B and other S100 proteins respond to calcium increases in the cell by coordinating calcium and undergoing a conformational change that allows them to interact with a variety of cellular targets. Although several three dimensional structures of S100 proteins are available in the calcium‐free (apo‐) state it has been observed that these structures appear to adopt a wide range of conformations in the EF2 site with respect to the positioning of helix III, the helix that undergoes the most dramatic calcium‐induced conformational change. In this work, we have determined the structure of human apo‐S100B at 10°C to examine whether temperature might be responsible for these structural differences. Further, we have used this data, and other available apo‐S100 structures, to show that despite the range of interhelical angles adopted in the apo‐S100 structures, normal Gaussian distributions about the mean angles found in the structure of human apo‐S100B are observed. This finding, only obvious from the analysis of all available apo‐S100 proteins, provides direct structural evidence that helix III is a loosely packed helix. This is likely a necessary functional property of the S100 proteins that facilitates the calcium‐induced conformational change of helix III. In contrast, the calcium‐bound structures of the S100 proteins show significantly smaller variability in the interhelical angles. This shows that calcium binding to the S100 proteins causes not only a conformational change but results in a tighter distribution of helices within the EF2 calcium binding site required for target protein interactions. Proteins 2008.

Rapid corepressor exchange from the trp-repressor/operator complex: An NMR study of [ul-13C/15N]-l-tryptophan

Summary[ul-13C/15N]-l-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (l-Trp) were unambiguously identified from gradient-enhanced 15N−1H HSQC, 13C−1H HSQC, 13C-and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4±0.52 s−1 at 45°C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or ‘breathing’ of the protein and/or DNA.

Solution structure of the Escherichia coli protein ydhR: A putative mono-oxygenase.

YdhR is a 101‐residue conserved protein from Escherichia coli. Sequence searches reveal that the protein has >50% identity to proteins found in a variety of other bacterial genomes. Using size exclusion chromatography and fluorescence spectroscopy, we determined that ydhR exists in a dimeric state with a dissociation constant of ∼40 nM. The three‐dimensional structure of dimeric ydhR was determined using NMR spectroscopy. A total of 3400 unambiguous NOEs, both manually and automatically assigned, were used for the structure calculation that was refined using an explicit hydration shell. A family of 20 structures was obtained with a backbone RMSD of 0.48 Å for elements of secondary structure. The structure reveals a dimeric α,β fold characteristic of the alpha+beta barrel superfamily of proteins. Bioinformatic approaches were used to show that ydhR likely belongs to a recently identified group of mono‐oxygenase proteins that includes ActVA‐Orf6 and YgiN and are involved in the oxygenation of polyaromatic ring compounds.

A re-examination of the structural and functional consequences of mutation of alanine-128 of the b subunit of Escherichia coli ATP synthase to aspartic acid

The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F(1) sector were analyzed. Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b(34-156). However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132. Unlike the wild-type sequence, the A128D mutant was unable to interact with F(1)-ATPase. These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F(1) but having little or no effect on the dimeric nature of b.