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D A Torchia

Publications and source records attributed to D A Torchia.

At least 55 records · Page 3Linked to original sources
PubMed1991

1H, 15N, and 13C NMR signal assignments of IIIGlc, a signal-transducing protein of Escherichia coli, using three-dimensional triple-resonance techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) of Escherichia coli. Virtually complete (98%) backbone 1H, 15N, and 13C nuclear magnetic resonance (NMR) signal assignments were determined by using a battery of triple-resonance three-dimensional (3D) NMR pulse sequences. In addition, nearly complete (1H, 95%; 13C, 85%) side-chain 1H and 13C signal assignments were obtained from an analysis of 3D 13C HCCH-COSY and HCCH-TOCSY spectra. These experiments rely almost exclusively upon one- and two-bond J couplings to transfer magnetization and to correlate proton and heteronuclear NMR signals. Hence, essentially complete signal assignments of this 168-residue protein were made without any assumptions regarding secondary structure and without the aid of a crystal structure, which is not yet available. Moreover, only three samples, one uniformly 15N-enriched, one uniformly 15N/13C-enriched, and one containing a few types of amino acids labeled with 15N and/or 13C, were needed to make the assignments. The backbone assignments together with the 3D 15N NOESY-HMQC and 13C NOESY-HMQC data have provided extensive information about the secondary structure of this protein [Pelton, J.G., Torchia, D.A., Meadow, N.D., Wong, C.-Y., & Roseman, S (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3479-3488]. The nearly complete set of backbone and side-chain atom assignments reported herein provide a basis for studies of the three-dimensional structure and dynamics of IIIGlc as well as its interactions with a variety of membrane and cytoplasmic proteins.

Amino Acid Sequence↗
PubMed1991

Deletion of the omega-loop in the active site of staphylococcal nuclease. 2. Effects on protein structure and dynamics.

It has been shown (Poole et al., 1991) that deletion of residues 44-49 from the sequence of staphylococcal nuclease (E43 SNase) results in an enzyme (E43 delta SNase) that is significantly more active than D43 SNase, an enzyme that differs from the wild-type enzyme by deletion of a single methylene group. In addition, both E43 delta SNase and D43 delta SNase are significantly more stable than their respective parent enzymes. Herein we use high-resolution 2D and 3D NMR spectroscopy to characterize the solution conformations of the four enzymes in order to better understand their differences in stability and activity. The backbone assignments of E43 SNase were extended to the three mutant proteins (uniformly 15N-enriched) by using 2D HSQC, 3D HOHAHA-HMQC, and 3D NOESY-HMQC spectra. The NOE patterns observed for E43 and D43 SNase in solution are consistent with the crystal structures of these proteins. The NOESY data further show that the intact and deleted proteins have essentially the same structures except that (a) the disordered omega-loops in the intact proteins are replaced by tight type II' turns, formed by residues 43-50-51-52, in the deleted proteins and (b) the orientation of the D43 side chain in crystalline D43 SNase differs from that found for D43 delta SNase in solution. Except for regions neighboring the omega-loops, the intact and deleted proteins show nearly identical amide 15N and 1H chemical shifts. In contrast, there are widespread, small and similar, chemical shift differences (a) between E43 SNase and D43 SNase and (b) between E43 delta SNase and D43 delta SNase.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1991

Secondary structure of the phosphocarrier protein IIIGlc, a signal-transducing protein from Escherichia coli, determined by heteronuclear three-dimensional NMR spectroscopy.

IIIGlc is a signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli. The secondary structure of IIIGlc is determined by heteronuclear (15N, 13C) three-dimensional NMR spectroscopy. Sequential, medium-range, and long-range nuclear Overhauser effects seen in NMR spectra are used to elucidate 11 antiparallel beta-strands and four helical segments. The medium-range nuclear Overhauser effect patterns suggest that the helices are either distorted alpha-helices or are of the 3(10) class. The amino acids separating the active-site histidine residues (His75 and His90) form two strands (Ala76-Ser81 and Val85-Phe91) of a six-stranded antiparallel beta-sheet that brings His90 and His75 in close proximity. Sequence similarities in IIIGlc and several other sugar-transport proteins suggest that the histidine residues within these proteins may be arranged in a similar manner. The 18-residue N-terminal peptide that precedes beta-strand Thr19-Ile22 in native IIIGlc is disordered and does not interact with the rest of the protein. Furthermore, removal of the N-terminal heptapeptide by a specific endopeptidase does not affect the structure of the remaining protein, thus explaining the phospho-acceptor activity of modified IIIGlc with the phospho-histidine-containing phosphocarrier protein of this system.

Amino Acid Sequence↗
PubMed1991

Complete 1H and 13C assignment of Lys and Leu sidechains of staphylococcal nuclease using HCCH-COSY and HCCH-TOCSY 3D NMR spectroscopy.

Complete proton and carbon sidechain assignments are reported for 22 lysine and 11 leucine residues in staphylococcal nuclease, an enzyme with 149 residues. These assignments are readily obtained in a direct manner from the correlations observed in the 3D HCCH-COSY and HCCH-TOCSY spectra and the known protein backbone assignments. These assignments open the way to detailed studies of the sidechain structure and dynamics at the active site, in the hydrophobic core and on the surface of the protein.

Carbon Isotopes↗
PubMed1991

Measurement of 15N-13C J couplings in staphylococcal nuclease.

15N-C alpha and 15N-C' J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with 15N and 13C. It is found that the 1JC'N coupling is similar for beta-sheet, J = 14.8 +/- 0.5 and for alpha-helix, J = 14.8 +/- 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J = 15.6 +/- 0.5). On average, 1JNC alpha are smaller for alpha-helical residues (J = 9.6 +/- 0.3 Hz) compared to beta-sheet (J = 10.9 +/- 0.8 Hz) and a substantial difference is observed for 2JNC alpha in alpha-helices (J = 6.4 +/- 0.4 Hz) and beta-sheets (J = 8.3 +/- 0.8 Hz).

Amino Acid Sequence↗
PubMed1990

An efficient NMR approach for obtaining sequence-specific resonance assignments of larger proteins based on multiple isotopic labeling.

By simultaneously incorporating in a protein 13C-carbonyl- and 15N-labeled amino acids with different levels of enrichment, characteristics asymmetric doublet-like patterns are observed for 15N nuclei that are directly adjacent to the 13C1-labeled residues, providing unambiguous identification of a large number of unique dipeptide fragments of the protein. Additional assignments and qualitative structural information can be obtained from such a selectively labeled protein by recording multiple bond correlation spectra. The procedure is demonstrated for the protein calmodulin, complexed with calcium.

Animals↗
PubMed1989

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease.

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.

Calcium↗
PubMed1989

Staphylococcal nuclease: sequential assignments and solution structure.

Sequential assignments are reported for backbone 15N and 1H of nearly all residues of staphylococcal nuclease (Nase) complexed with thymidine 3',5'-diphosphate and Ca2+. Because of the relatively large size of the Nase ternary complex, Mr 18K, the crucial element of our assignment strategy was the use of isotope-edited two-dimensional NMR spectra, particularly 15N-edited nuclear Overhauser enhancement spectroscopy (NOESY), 15N-edited J-correlated spectroscopy (COSY), and 1H/15N or 1H/13C heteronuclear multiple quantum shift correlation spectroscopy (HMQC). These experiments, together with the more conventional NOESY, COSY, and homonuclear Hartmann-Hahn spectra of natural abundance or deuteriated samples, yielded backbone assignments of 127 of the 136 residues in the structured part of the protein. Using the NOESY data, we identified three helical domains and several beta-sheets which were in close correspondence with secondary structure identified in the crystal structure. Moreover, many long-range NOESY connectivities were identified that were in agreement with distances derived from the crystal structure. The region of the sequence in the neighborhood of residue 50 appears to be more flexible and disordered in solution than in the crystal. Very slowly exchanging amide protons are those found to be hydrogen bonded in the crystal structure; however, even hydrogen-bonded amides located within similar types of regular secondary structures, e.g., alpha-helices, exchange with greatly different rates.

Amino Acid Sequence↗
PubMed1988

Solid-state NMR studies of the dynamics and structure of mouse keratin intermediate filaments.

The molecular dynamics and structural organization of mouse epidermal keratin intermediate filaments (IF) have been studied via solid-state nuclear magnetic resonance (NMR) experiments performed on IF labeled both in vivo and in vitro with isotopically enriched amino acids. As a probe of the organization of the peripheral glycine-rich end domains of the IF, carbon-13 NMR experiments have been performed on subfilamentous forms (prekeratin) and on IF reassembled in vitro that had been labeled with either [1-13C]glycine or [2-13C]glycine, as more than 90% of the glycines of the keratins are located in the end domains. Although cross-labeling to seryl residues was observed, the proportion of serine located in the end domains is nearly the same as that for glycine. Measurements of carbon relaxation times, nuclear Overhauser enhancements, and signal intensities show that the motions of the peptide backbone in the end domains are effectively isotropic, with average correlation times distributed over the range of 0.2-20 ns. These results indicate that the end domains of IF are remarkably flexible and have little or no structural order. To probe the structural organization of the coiled-coil rod domains of the IF, separate samples of native keratin IF, raised in primary tissue culture, were labeled with L-[1-13C]leucine, L-[2H10]leucine, or L-[2,3,3-2H3]leucine, as greater than 90% of the leucyl residues of the keratin IF types studied are located in the coiled coils which form the central core of IF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

NMR signal assignments of amide protons in the alpha-helical domains of staphylococcal nuclease.

We report complete assignments of the amide proton signals in the three long dNN connectivity sequences observed in the NOESY spectrum of deuteriated staphylococcal nuclease (Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+, Mr 18K. The assignments are made by comparing NOESY spectra with 1H-15N and 1H-13C heteronuclear multiple-quantum shift correlation (HMQC) spectra of Nase samples containing 15N- and 13C-labeled amino acids. The assignments show that the residues which are linked by the dNN connectivity sequences are located in three alpha-helical domains of Nase. Our results indicate that by combining NOESY and HMQC spectra of appropriately labeled samples it should be possible to delineate and study alpha-helical domains in soluble proteins having molecular weights that are greater than 18K.

Amides↗
PubMed1988

Comparison of the solution and crystal structures of staphylococcal nuclease with 13C and 15N chemical shifts used as structural fingerprints.

We report high-resolution 13C and 15N NMR spectra of crystalline staphylococcal nuclease (Nase) complexed to thymidine 3',5'-diphosphate and Ca2+. High sensitivity and resolution are obtained by applying solid-state NMR techniques--high power proton decoupling and cross-polarization magic angle sample spinning (CPMASS)--to protein samples that have been efficiently synthesized and labeled by an overproducing strain of Escherichia coli. A comparison of CPMASS and solution spectra of Nase labeled with either [methyl-13C]methionine or [15N]valine shows that the chemical shifts in the crystalline and solution states are virtually identical. This result is strong evidence that the protein conformations in the solution and crystalline states are nearly the same. Because of the close correspondence of the crystal and solution chemical shifts, sequential assignments obtained in solution apply to the crystal spectra. It should therefore be possible to study the molecular structure and dynamics of many sequentially assigned atomic sites in Nase crystals. Similar experiments are applicable to the growing number of proteins that can be obtained from efficient expression systems.

Carbon Isotopes↗
PubMed1987

Molecular dynamics of collagen side chains in hard and soft tissues. A multinuclear magnetic resonance study.

We have prepared samples of (a) intact calvaria collagen (cross-linked and mineralized), (b) intact tendon collagen (cross-linked but not mineralized), and (c) reconstituted chick calvaria collagen (not cross-linked and not mineralized) containing [methyl-2H3]methionyl, [4,4-2H2]pyrrolidinyl, (4-fluorophenyl)alanyl, and [6-15N]lysyl residues. Using multinuclear magnetic resonance spectroscopy, we have investigated the molecular dynamics of the labeled amino acids. Guided by model compound studies, we reached the following conclusions regarding collagen side chain dynamics from our analysis of line shapes and relaxation rates. At 22 degrees C, imino residues in all samples have flexible rings with root mean square angular fluctuations in the 11-30 degree range. Nearly all labeled amino acid side chains reorient about at least two side chain single bonds. At temperatures below -30 degrees C, most of these side chain motions are absent in all the samples. Surprisingly, in contrast with results obtained for backbone motions, side chain motions are only marginally more hindered in mineralized samples as compared with nonmineralized samples, a result we discuss with reference to collagen-mineral interactions. We also discuss the possible relationship between collagen dynamics and function.

Achilles Tendon↗
PubMed1986

A multinuclear NMR study of the interactions of cations with proteoglycans, heparin, and Ficoll.

Measurements of NMR relaxation rates of 23Na, 39K, 25Mg and 43Ca were used to evaluate the extent of cation binding in solution to bovine nasal cartilage proteoglycans, hog mucosal heparin, and Ficoll (Pharmacia). The two most important factors determining relaxation rates in the presence of the polymers examined were polymer concentration and charge density. We found that proteoglycans did not bind monovalent cations but did bind divalent cations to a relatively small extent. Heparin bound monovalent and divalent cations to a much greater extent. Assembly of glycosaminoglycan chains into proteoglycan aggregates had no effect on the extent of cation binding.

Animals↗
PubMed1985

Nanosecond fluctuations of the molecular backbone of collagen in hard and soft tissues: a carbon-13 nuclear magnetic resonance relaxation study.

We have determined the amplitude of nanosecond fluctuations of the collagen azimuthal orientation in intact tissues and reconstituted fibers from an analysis of 13C NMR relaxation data. We have labeled intact rat calvaria and tibia collagen (mineralized and cross-linked), intact rat tail tendon and demineralized bone collagen (cross-linked), and reconstituted lathyritic (non-cross-linked) chick calvaria collagen with [2-13C]glycine. This label was chosen because one-third of the amino acid residues in collagen are glycine and because the 1H-13C dipolar coupling is the dominant relaxation mechanism. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancements were measured at 15.09 and 62.98 MHz at 22 and -35 degrees C. The measured NMR parameters have been analyzed by using a dynamic model in which the azimuthal orientation of the molecule fluctuates as a consequence of reorientation about the axis of the triple helix. We have shown that if root mean square fluctuations in the azimuthal orientations are small, gamma rms much less than 1 rad, the correlation function decays with a single correlation time tau and T1 depends only upon tau and gamma rms and not the detailed model of motion. Our analysis shows that, at 22 degrees C, tau is in the 1-5-ns range for all samples and gamma rms is 10 degrees, 9 degrees, and 5.5 degrees for the non-cross-linked, cross-linked, and mineralized samples, respectively. At -35 degrees C, gamma rms is less than 3 degrees for all samples. These results show that mineral and low temperature significantly restrict the amplitude of nanosecond motions of the collagen backbone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Detection of cis and trans X-Pro peptide bonds in proteins by 13C NMR: application to collagen.

Heretofore the complexity of natural abundance spectra has precluded the use of 13C NMR to detect cis peptide bonds in proteins. We have incorporated [4-13C]proline into chicken calvaria collagen and report here well-resolved C gamma signals, arising from cis and trans X-Pro and X-Hyp peptide bonds (where X is any amino acid residue) in the 13C NMR spectrum of the thermally unfolded protein. Measurement of 13C signal areas shows that 16% of the X-Pro and 8% of X-Hyp bonds are cis in the unfolded collagen. These results strongly support the conclusion drawn from kinetic studies that cis-trans isomerization of peptide bonds is the rate-limiting step in helix propagation after nucleation. Our method can be applied to other proteins as well and should aid in testing the generality of the hypothesis of Brandts, Halvorson, and Brennan that cis-trans isomerization is the rate-limiting step in protein folding when proline is present.

Animals↗
PubMed1984

Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis.

High-resolution 31P nuclear magnetic resonance spectroscopy and 14C fluorography have been used to identify and quantitate intermediates of the Embden-Meyerhof pathway in intact cells and cell extracts of Streptococcus lactis. Glycolysing cells contained high levels of fructose 1,6-bisphosphate (a positive effector of pyruvate kinase) but comparatively low concentrations of other glycolytic metabolites. By contrast, starved organisms contained only high levels of 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate. The concentration of Pi (a negative effector of pyruvate kinase) in starved cells was fourfold greater than that maintained by glycolysing cells. The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruvate kinase: the increase in the phosphoenolpyruvate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi (by a maltose-plus-arginine phosphate trap) caused the restoration of pyruvate kinase activity in starved cells. Time course studies showed that Pi was conserved by formation of fructose 1,6-bisphosphate during glycolysis. Conversely, during starvation high levels of Pi were generated concomitant with depletion of intracellular fructose 1,6-bisphosphate. The concentrations of Pi and fructose 1,6-bisphosphate present in starved and glycolysing cells of S. lactis varied inversely. The activity of pyruvate kinase in the growing cell may be modulated by the relative concentrations of the two antagonistic effectors.

Carbon Radioisotopes↗