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G M Clore

Publications and source records attributed to G M Clore.

At least 145 records · Page 8Linked to original sources
PubMed1992

1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain.

The structure of the B2 immunoglobulin-binding domain of streptococcal protein G has been determined at 1.67-A resolution using a combination of single isomorphous replacement (SIR) phasing and manual fitting of the coordinates of the NMR structure of B1 domain of streptococcal protein G [Gronenborn, A. M., et al. (1991) Science 253, 657-661]. The final R value was 0.191 for data between 8.0 and 1.67 A. The structure described here has 13 residues preceding the 57-residue Ig-binding domain and 13 additional residues following it, for a total of 83 residues. The 57-residue binding domain is well-determined in the structure, having an average B factor of 18.0. Only residues 8-77 could be located in the electron density maps, with the ends of the structure fading into disorder. Like the B1 domain, the B2 domain consists of four beta-strands and a single helix lying diagonally across the beta-sheet, with a -1, +3 chi, -1 topology. This small structure is extensively hydrogen-bonded and has a relatively large hydrophobic core. These structural observations may account for the exceptional stability of protein G. A comparison of the B2 domain X-ray structure and the B1 domain NMR structure showed minor differences in the turn between strands and two and a slight displacement of the helix relative to the sheet. Hydrogen bonds between crystallographically related molecules account for most of these differences.

Amino Acid Sequence↗
PubMed1992

Analysis of the backbone dynamics of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase using 15N relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled ribonuclease H (RNase H) domain of human immunodeficiency virus (HIV-1) reverse transcriptase have been investigated using two-dimensional inverse-detected heteronuclear 15N-1HNMR spectroscopy. 15N T1, T2, and nuclear Overhauser enhancement (NOE) data were obtained for 107 out of a total of 134 backbone amide groups. The overall rotational correlation time (tau R) for the protein at 26 degrees C is 10.4 ns. The backbone N-H vectors for all the measurable residues exhibit very fast motions on a time scale of less than or equal to 20 ps. The 15N relaxation data for only 14 residues can be explained by this single internal motion alone. A further 39 residues display a second motion on a time scale ranging from 28.8 ps to 3.9 ns, while another 15 residues are characterized by an additional motion on the 170-ns to 2.25-ms time scale resulting in 15N T2 exchange line broadening. There are 39 residues that exhibit both the additional 15N T2 exchange line broadening and the slow (28.8 ps-3.9 ns) internal motion. Thus, the RNase H domain experiences extensive mobility throughout its structure as evidenced by the 93 residues which exhibit multiple modes of motion. Distinctly mobile regions of the protein are identified by large decreases in the overall order parameter (S2) and correspond to the C-terminal residues and the loop regions between beta-strands beta 1 and beta 2 and between alpha-helix alpha B and beta-strand beta 4.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV Reverse Transcriptase↗
PubMed1992

Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu.

Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue. The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected. The modified peptide was unstable under mildly acid or mildly basic conditions. Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue. Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography. Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively. Given the instability of the modification, it may be more prevalent than recognized hitherto. Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way.

Amino Acid Sequence↗
PubMed1992

Combining experimental information from crystal and solution studies: joint X-ray and NMR refinement.

Joint refinement of macromolecules against crystallographic and nuclear magnetic resonance (NMR) observations is presented as a way of combining experimental information from the two methods. The model of interleukin-1 beta derived by the joint x-ray and NMR refinement is shown to be consistent with the experimental observations of both methods and to have crystallographic R value and geometrical parameters that are of the same quality as or better than those of models obtained by conventional crystallographic studies. The few NMR observations that are violated by the model serve as an indicator for genuine differences between the crystal and solution structures. The joint x-ray-NMR refinement can resolve structural ambiguities encountered in studies of multidomain proteins, in which low- to medium-resolution diffraction data can be complemented by higher resolution NMR data obtained for the individual domains.

Amino Acid Sequence↗
PubMed1992

A 500 ps molecular dynamics simulation study of interleukin-1 beta in water. Correlation with nuclear magnetic resonance spectroscopy and crystallography.

We report the results of a 500 ps molecular dynamics simulation of the cytokine interleukin-1 beta, a protein of 153 amino acids, immersed in a sphere of 3783 bulk water molecules with a radius of 33 A. The simulation reproduces the amplitudes of the fast librational motions of the backbone N-H bonds determined from 15N nuclear magnetic relaxation data, as well as the crystallographic B-factors. Moreover, this study suggests a molecular picture of the nature of the slow internal motions that have been inferred from nuclear magnetic resonance relaxation experiments. These experiments indicated that, in addition to fast motions common to all residues, 32 surface residues exhibit slow motions on the 400 ps to 5 ns time-scale. While the present simulation is not sufficiently long to provide a quantitative description of events on this time-scale, it is long enough to observe several large amplitude transitions that are likely candidates for these slow motions. Specifically, in many of these 32 residues, the N-H groups are hydrogen bonded and infrequent dihedral transitions cause the N-H vectors to jump between states with well-defined orientations. It is shown that the time course of the angular reorientational correlation functions of these residues calculated from the trajectory is a reflection of the random times at which these infrequent jumps happen to have occurred. Thus, while the rate of these transitions cannot be quantified, the simulated decay of these correlation functions is completely consistent with the physical picture in which the N-H vectors, in addition to fast librational motion, undergo large amplitude jumps between conformations stabilized by hydrogen bonds.

Computer Simulation↗
PubMed1992

Three-dimensional solution structure of human interleukin-4 by multidimensional heteronuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of recombinant human interleukin-4, a protein of 133 residues and 15.4 kilodaltons that plays a key role in the immune and inflammatory systems, has been solved by multidimensional heteronuclear magnetic resonance spectroscopy. The structure 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 overall topology is remarkably similar to that of growth hormone and granulocyte-macrophage colony stimulating factor, despite the absence of any sequence homology, and substantial differences in the relative lengths of the helices, the length and nature of the various connecting elements, and the pattern of disulfide bridges. These three proteins, however, bind to cell surface receptors belonging to the same hematopoietic superfamily, which suggests that interleukin-4 may interact with its receptor in an analogous manner to that observed in the crystal structure of the growth hormone-extracellular receptor complex.

Amino Acid Sequence↗
PubMed1992

1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i + 1),NH(i + 1) correlations are observed in the HNCO experiment, the C alpha H(i), C alpha (i)-N(i + 1) correlations in the HCA(CO)N experiment, the C alpha(i)-N(i + 1),NH(i + 1) correlations in the HNCA and HN(CO)CA experiments, the C alpha H(i)-N(i + 1),NH(i + 1) correlations in the H(CA)NH and HN(CO)HB experiments, and the C beta H(i)-N(i + 1),NH(i + 1) correlations in the HN(CO)HB experiments. The backbone intraresidue C alpha H(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C beta H(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13C alpha(i)-15N(i)-NH(i) correlations by the HNCA experiment, and the C alpha H(i)-13C alpha(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.

Amino Acid Sequence↗
PubMed1992

Determination of the secondary structure and folding topology of human interleukin-4 using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure of human recombinant interleukin-4 (IL-4) has been investigated by three-dimensional (3D) 15N- and 13C-edited nuclear Overhauser (NOE) spectroscopy on the basis of the 1H, 15N, and 13C assignments presented in the preceding paper [Powers, R., Garrett, D. S., March, C. J., Frieden, E. A., Gronenborn, A. M., & Clore, G. M. (1992) Biochemistry (preceding paper in this issue)]. Based on the NOE data involving the NH, C alpha H, and C beta H protons, as well as 3JHN alpha coupling constant, amide exchange, and 13C alpha and 13C beta secondary chemical shift data, it is shown that IL-4 consists of four long helices (residues 9-21, 45-64, 74-96, and 113-129), two small helical turns (residues 27-29 and 67-70), and a mini antiparallel beta-sheet (residues 32-34 and 110-112). In addition, the topological arrangement of the helices and the global fold could be readily deduced from a number of long-range interhelical NOEs identified in the 3D 13C-edited NOE spectrum in combination with the spatial restrictions imposed by three disulfide bridges. These data indicate that the helices of interleukin-4 are arranged in a left-handed four-helix bundle with two overhand connections.

Amino Acid Sequence↗
PubMed1992

Solution structure of a calmodulin-target peptide complex by multidimensional NMR.

The three-dimensional solution structure of the complex between calcium-bound calmodulin (Ca(2+)-CaM) and a 26-residue synthetic peptide comprising the CaM binding domain (residues 577 to 602) of skeletal muscle myosin light chain kinase, has been determined using multidimensional heteronuclear filtered and separated nuclear magnetic resonance spectroscopy. The two domains of CaM (residues 6 to 73 and 83 to 146) remain essentially unchanged upon complexation. The long central helix (residues 65 to 93), however, which connects the two domains in the crystal structure of Ca(2+)-CaM, is disrupted into two helices connected by a long flexible loop (residues 74 to 82), thereby enabling the two domains to clamp residues 3 to 21 of the bound peptide, which adopt a helical conformation. The overall structure of the complex is globular, approximating an ellipsoid of dimensions 47 by 32 by 30 angstroms. The helical peptide is located in a hydrophobic channel that passes through the center of the ellipsoid at an angle of approximately 45 degrees with its long axis. The complex is mainly stabilized by hydrophobic interactions which, from the CaM side, involve an unusually large number of methionines. Key residues of the peptide are Trp4 and Phe17, which serve to anchor the amino- and carboxyl-terminal halves of the peptide to the carboxyl- and amino-terminal domains of CaM, respectively. Sequence comparisons indicate that a number of peptides that bind CaM with high affinity share this common feature containing either aromatic residues or long-chain hydrophobic ones separated by a stretch of 12 residues, suggesting that they interact with CaM in a similar manner.

Amino Acid Sequence↗
PubMed1992

High-resolution solution structure of the double Cys2His2 zinc finger from the human enhancer binding protein MBP-1.

The high-resolution three-dimensional structure of a synthetic 57-residue peptide comprising the double zinc finger of the human enhancer binding protein MBP-1 has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1280 experimental restraints. A total of 30 simulated annealing structures were calculated. The backbone atomic root-mean-square distributions about the mean coordinate positions are 0.32 and 0.33 A for the N- and C-terminal fingers, respectively, and the corresponding values for all atoms, excluding disordered surface side chains, are 0.36 and 0.40 A. Each finger comprises an irregular antiparallel sheet and a helix, with the zinc tetrahedrally coordinated to two cysteines and two histidines. The overall structure is nonglobular in nature, and the angle between the long axes of the helices is 47 +/- 5 degrees. The long axis of the antiparallel sheet in the N-terminal finger is approximately parallel to that of the helix in the C-terminal finger. Comparison of this structure with the X-ray structure of the Zif-268 triple finger complexed with DNA indicates that the relative orientation of the individual zinc fingers is clearly distinct in the two cases. This difference can be attributed to the presence of a long Lys side chain in the C-terminal finger of MBP-1 at position 40, instead of a short Ala or Ser side chain at the equivalent position in Zif-268. This finding suggests that different contacts may be involved in the binding of the zinc fingers of MBP-1 and Zif-268 to DNA, consistent with the findings from methylation interference experiments that the two fingers of MBP-1 contact 10 base pairs, while the three fingers of Zif-268 contact only 9 base pairs.

Amino Acid Sequence↗
PubMed1992

Reversible steps in the bacteriorhodopsin photocycle.

The absorbance changes that accompany the light-driven proton-pumping cycle of bacteriorhodopsin measured over a broad range of times, wavelengths, temperatures, and pH values have been globally fitted to the kinetic model K in equilibrium with L in equilibrium with X in equilibrium with M in equilibrium with N in equilibrium with O----bR. A remarkably good fit to the data was obtained by optimizing the rate constants at 20 degrees C and the corresponding activation energies at each pH value, together with the extinction coefficients for each intermediate, which were assumed to be independent of both pH and temperature. Back-reactions are included for all but the last step of the cycle and are found to be essential for fitting the data. The rates of these reactions are large, and the analogous irreversible model produced significantly worse fits to the data. Small systematic differences between the fit and the experimental data associated with the X, M, and O intermediates, together with the inability of the model to produce spectra for the X and M intermediates consistent with their assignment as molecular species, indicate that this model must be an incomplete description of the photocycle. We suggest that these problems arise from the presence of additional occupied states that are difficult to distinguish on the basis of their visible absorption spectra alone.

Bacteriorhodopsins↗
PubMed1992

Relationship between electrostatics and redox function in human thioredoxin: characterization of pH titration shifts using two-dimensional homo- and heteronuclear NMR.

The electrostatic behavior of potentially titrating groups in reduced human thioredoxin was investigated using two-dimensional (2D) 1H and 15N nuclear magnetic resonance (NMR) spectroscopy. A total of 241 chemical shift titration curves were measured over the pH range of 2.1-10.6 from homonuclear 1H-1H Hartmann-Hahn (HOHAHA) and heteronuclear 1H-15N Overbodenhausen correlation spectra. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the determination of pKas for certain isolated ionizable groups, including the single histidine residue at position 43 (pKa = 5.5 +/- 0.1) and a number of aspartic and glutamic acid carboxylate groups. Many of the titration curves demonstrate complex behavior due to the effects of interacting titrating groups, the long range of electrostatic interactions through the protein interior, and, perhaps, pH-induced conformational changes on the chemical shifts. Unambiguous assignment of the pKas for most of the 38 potentially ionizing groups of human thioredoxin could therefore not be made. In addition, there was no clear evidence that Asp-26 titrates in a manner corresponding to that observed in the Escherichia coli protein [Dyson, H. J., Tennant, L. L., & Holmgren, A. (1991) Biochemistry 30, 4262-4268]. The pKas of the active site cysteines were measured, however, with Cys-32 having an anomalously low value of 6.3 +/- 0.1 and that of Cys-35 between 7.5 and 8.6. These pKas are in agreement with proposed mechanisms for redox catalysis of thioredoxin and previously measured pKas within the active site of E. coli thioredoxin [Kallis, G. B., & Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265]. The stabilization of a thiolate anion at physiological pH can be explained by the interaction of the S gamma of Cys-32 with the amide of Cys-35 observed in the previously determined high-resolution solution structure of reduced human thioredoxin [Forman-Kay, J. D., Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2685-2698].

Binding Sites↗
PubMed1992

Three-dimensional solution structure of the E3-binding domain of the dihydrolipoamide succinyltransferase core from the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

The three-dimensional solution structure of a 51-residue synthetic peptide comprising the dihydrolipoamide dehydrogenase (E3)-binding domain of the dihydrolipoamide succinyltransferase (E2) core of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli has been determined by nuclear magnetic resonance spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure is based on 630 approximate interproton distance and 101 torsion angle (phi, psi, chi 1) restraints. A total of 56 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions for residues 12-48 of the synthetic peptide is 1.24 A for the backbone atoms, 1.68 A for all atoms, and 1.33 A for all atoms excluding the six side chains which are disordered at chi 1 and the seven which are disordered at chi 2; when the irregular partially disordered loop from residues 31 to 39 is excluded, the rms distribution drops to 0.77 A for the backbone atoms, 1.55 A for all atoms, and 0.89 A for ordered side chains. Although proton resonance assignments for the N-terminal 11 residues and the C-terminal 3 residues were obtained, these two segments of the polypeptide are disordered in solution as evidenced by the absence of nonsequential nuclear Overhauser effects. The solution structure of the E3-binding domain consists of two parallel helices (residues 14-23 and 40-48), a short extended strand (24-26), a five-residue helical-like turn, and an irregular (and more disordered) loop (residues 31-39). This report presents the first structure of an E3-binding domain from a 2-oxo acid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗
PubMed1992

Localization of bound water in the solution structure of the immunoglobulin binding domain of streptococcal protein G. Evidence for solvent-induced helical distortion in solution.

The presence of bound water in the solution structure of the IgG binding domain of streptococcal protein G has been investigated by nuclear magnetic resonance using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. The backbone amide protons of three residues, Ala20, Gln32 and Tyr33, are found to be in close proximity to bound water. Examination of the three-dimensional structure of the IgG binding domain indicates that in the vicinity of these three residues there are no backbone groups that do not already participate in hydrogen bonding and there are no suitably placed side-chain groups available for hydrogen bonding with water. As the lifetime of the bound water detected in this nuclear magnetic resonance experiment is greater than about one nanosecond, it is likely that the two bound water molecules participate in a bifurcating hydrogen bonding network comprising a CO-NH hydrogen bonded pair, such that the water molecule accepts a hydrogen bond from the NH proton and donates one to the carbonyl oxygen with the result that the amide proton is involved in a three center hydrogen bond. On the basis of the structure, one water molecule participates in such an interaction with the Ala20(NH)-Met1(CO) hydrogen bonded pair at the beginning of an anti-parallel beta-sheet, and the other with the Tyr33(NH)-Val29(CO) hydrogen bonded pair in the single alpha-helix. The latter, which is external and solvent accessible, is associated with a distortion in the alpha-helix centered around Tyr33 which consists of a significant increase in the CO(i-4)-N(i) and CO(i-4)-NH(i) distances relative to those in the rest of the helix, as well as a significant departure in the phi, psi angles of Tyr33 relative to regular helical geometry. Such solvent induced distortions in alpha-helices have been previously noticed in crystal structures and were postulated as possible folding intermediates for helical structures. The present observation of this phenomenon in solution indicates, however, that these water molecules are tightly bound and represent an integral part of the protein framework.

Bacterial Proteins↗
PubMed1992

1H and 15N resonance assignments and secondary structure of the human thioredoxin C62A, C69A, C73A mutant.

The complete assignment of 1H and 15N backbone resonances and near-complete 1H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional 15N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay, J.D. et al. (1991) Biochemistry, 30, 2685-2698]. An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.

Amino Acid Sequence↗
PubMed1991

Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy.

A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein.

Amino Acid Sequence↗
PubMed1991

Secondary structure of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase in solution using three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy.

The solution structure of the ribonuclease H domain of HIV-1 reverse transcriptase has been investigated by three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. The domain studied has 138 residues and comprises residues 427 to 560 of the 66 kDa reverse transcriptase with an additional four residues at the N terminus. Initial studies on the wild-type protein were hindered by severe differential line broadening, presumably due to conformational averaging. Mutation of the single tryptophan residue located in a loop at position 113 (position 535 in the reverse transcriptase sequence) to an alanine resulted in much improved spectral properties with no apparent change in structure. 1H, 15N and 13C backbone resonances were assigned sequentially using a range of three-dimensional double and triple resonance heteronuclear experiments on samples of uniformly (greater than 95%) 15N and 15N/13C-labeled protein, and the secondary structure was elucidated from a qualitative analysis of data derived from three-dimensional 15N- and 13C-edited nuclear Overhauser enhancement spectra. The secondary structure comprises three alpha-helices and five strands arranged in a mixed parallel/antiparallel beta-sheet with a +1, +1, -3x, -1x topology. The C-terminal region from residue 114 onwards appears to be conformationally disordered in solution as evidenced by an almost complete absence of sequential and medium range nuclear Overhauser effects.

Amino Acid Sequence↗