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D A Torchia

Publications and source records attributed to D A Torchia.

At least 19 recordsLinked to original sources
PubMed1992

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Leucine↗
PubMed1992

Structural comparison of phosphorylated and unphosphorylated forms of IIIGlc, a signal-transducing protein from Escherichia coli, using three-dimensional NMR techniques.

The 18.1-kDa protein IIIGlc from Escherichia coli acts as both a phosphocarrier protein in the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and as a signal-transducing protein with respect to the uptake of non-PTS sugars. Phosphorylation of IIIGlc at the N epsilon (N3) position of His-90 was effected through a regeneration system that included MgCl2, DTT, excess PEP, and catalytic amounts of Enzyme I and HPr. NH, 15N, and 13C alpha signal assignments for P-IIIGlc were made through comparison of 15N-1H correlation spectra (HSQC) of uniformly 15N-labeled preparations of phosphorylated and unphosphorylated protein and through analysis of three-dimensional triple-resonance HNCA spectra of P-IIIGlc uniformly labeled with both 15N and 13C. Backbone and side-chain 1H and 13C beta signals were assigned using 3D heteronuclear HCCH-COSY and HCCH-TOCSY spectra of P-IIIGlc. Using this approach, the assignments were made without reference to nuclear Overhauser effect data or assumptions regarding protein structure. The majority of NH, 15N, H alpha, and 13C alpha chemical shifts measured for P-IIIGlc were identical to those obtained for the unphosphorylated protein [Pelton, J. G., Torchia, D. A., Meadow, N. D., Wong, C.-Y., & Roseman, S. (1991) Biochemistry 30, 10043]. Those signals that exhibited shifts corresponded to residues within four segments (1) Leu-87-Gly-100, (2) Val-36-Val-46, (3) His-75-Ser-78, and (4) Ala-131-Val-138. These four segments are in close proximity to the active site residues His-75 and His-90 in the unphosphorylated protein [Worthylake, D., Meadow, N. D., Roseman, S., Liao, D., Hertzberg, O., & Remington, S.J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10382], and the chemical shift data provide strong evidence that if any structural changes accompany phosphorylation, they are confined to residues in these four segments. This conclusion is confirmed by comparing NOEs observed in 3D 15N/13C NOESY-HMQC spectra of the two forms of the protein. No NOE differences are seen for residues having the same chemical shifts in IIIGlc and P-IIIGlc. Furthermore, with the exception of residues Ala-76, Asp-94, and Val-96, the NOEs of residues (in the four segments) which exhibited chemical shift differences also had the same NOEs in IIIGlc and P-IIIGlc. In the case of residues Ala-76, Asp-94, and Val-96, minor differences in NOEs, corresponding to interproton distances changes of less than 1.5 A, were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
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↗
PubMed1979

13C NMR quantitation of polymer in deoxyhemoglobin S gels.

13C/1H magnetic double-resonance spectroscopy has been used to quantitate the amount of polymerized hemoglobin S in deoxygenated gels at 30 degrees C, for samples whose hemoglobin concentration range from 21 to 32 g/dl. Scalar- and dipolar-decoupled spectra and a 13C proton-enhanced dipolar-decoupled spectrum were recorded for each sample as was a scalar-decoupled spectrum for a matching oxyhemoglobin S control. The difference between the oxyhemoglobin S and deoxyhemoglobin S scalar-decoupled spectra was used to determine the polymer fraction, and this value was compared with the polymer fraction determined by using ultracentrifugation sedimentation on the same sample (assuming a two-phase model). The polymer fraction value determined by uncorrected sedimentation averaged 0.15 more than the value obtained from NMR. The discrepancy between the two techniques was largely removed when the analysis of the sedimentation data included a correction for depletion of hemoglobin in the supernatant or sol phase due to sedimentation of free molecules. The best fit to both the sedimentation and NMR data was obtained by using a solubility of deoxyhemoglobin S at 30 degrees C of 17.3 +/- 1 g/dl. These results indicate that the NMR techniques, which do not require separation of the sample into a sol phase and a pellet phase, provide quantitative information about the deoxyhemoglobin S polymer and will be useful for studies of sickle erythrocytes.

Erythrocytes↗
PubMed1979

Enamel matrix: structural proteins.

Cell-free, fetal bovine enamel tissue was examined intact by high resolution. 13C Fourier transform, nuclear magnetic resonance spectroscopy. Two types of protein chains were observed under these conditions, one exhibiting rapid mobility and accounting for approximately two-thirds of the enamel matrix, while the other exhibited restricted or anisotropic segmental motion and accounted for the remaining third of the matrix. Sequential extraction of this fetal enamel under non-degradative conditions with dissociative solvents yielded two biochemically distinct populations of matrix protein. As expected, the bulk of the matrix consisted of proline-rich amelogenins, although the SDS-gel electrophoresis molecular weights for these proteins were somewhat higher than those reported using other extraction methods. Approximately fifteen percent of the total matrix consisted of much higher molecular weight phosphoproteins (46,000-72,000 daltons) whose amino acid composition closely resembled that reported for mature enamel protein. These high molecular weight proteins were tightly bound to the fetal enamel apatite crystallites.

Animals↗