Biomedical subjects

A Bax

Publications and source records attributed to A Bax.

At least 91 records · Page 5Linked to original sources

PubMed1993

Minimizing the effects of radio-frequency heating in multidimensional NMR experiments.

Application of radio-frequency power in multidimensional NMR experiments can significantly increase the sample temperature compared to that of the surrounding gas flow. Radio-frequency heating effects become more severe at higher magnetic field strengths and ionic strengths. The effects are particularly noticeable for experiments that utilize 1H and/or 13C isotropic mixing and broadband decoupling. If radio-frequency power is applied during the systematically increasing evolution period t1, the sample temperature can change with t1 and thereby cause line-shape distortions. Such distortions are easily avoided by ensuring that the average radio-frequency power remains constant during the entire experiment.

Carbon Isotopes↗

PubMed1993

Measurement of two- and three-bond 13C-1H J couplings to the C delta carbons of leucine residues in staphylococcal nuclease.

A new 1H-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond 13C-1H couplings in proteins with selectively 13C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 13C]-labeled in all 11 leucine positions and ligated with thymidine 3',5'-biphosphate and Ca2+. Two- and three-bond 13C methyl-proton couplings are reported and, together with the measured three-bond JC alpha C delta in uniformly 13C-enriched staphylococcal nuclease, the chi 2-angles and the stereospecific assignments of the C delta methyl group with respect to the prochiral beta-protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of 13C relaxation times have measured coupling constants that are indicative of motional averaging.

Amino Acid Sequence↗

PubMed1993

The use of 1JC alpha H alpha coupling constants as a probe for protein backbone conformation.

Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (+/- 0.5 Hz) measurement of one bond JC alpha H alpha coupling constants in proteins that are uniformly enriched with 13C. An empirical phi,psi-surface is calculated which describes the deviation of 1JC alpha H alpha from its random coil value, using 203 1JC alpha H alpha values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which phi and psi are known with good precision from previous X-ray crystallographic studies. Residues in alpha-helical conformation exhibit positive deviations of 4-5 Hz, whereas deviations in beta-sheet are small and, on average, slightly negative. Data indicate that 1JC alpha H alpha depends primarily on psi, and that 1JC alpha H alpha may be useful as a qualitative probe for secondary structure. Comparison of 1JC alpha H alpha coupling constants measured in free calmodulin and in its complex with a 26-amino-acid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the 'central helix' is even more extensive than in free calmodulin.

Calmodulin↗

PubMed1993

Isotope-edited multidimensional NMR of calcineurin B in the presence of the non-deuterated detergent CHAPS.

At the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of approximately 25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to 15N or 13C are removed by isotope filtering and purge pulses.

Calcineurin↗

PubMed1992

1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma.

1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.

Amino Acid Sequence↗

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

Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible.

The backbone dynamics of Ca(2+)-saturated recombinant Drosophila calmodulin has been studied by 15N longitudinal and transverse relaxation experiments, combined with 15N(1H) NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.5-0.6 range. The anisotropy observed in the motion of the two globular calmodulin domains is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that, for the purposes of 15N relaxation, the tumbling of the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of calmodulin is effectively independent. A slightly shorter motional correlation time (tau c approximately 6.3 ns) is obtained for the C-terminal domain compared to the N-terminal domain (tau c approximately 7.1 ns), in agreement with the smaller size of the C-terminal domain. A high degree of mobility, with order parameters of approximately 0.5, is also observed in the loop that connects the first with the second EF-hand type calcium binding domain and in the loop connecting the third and fourth calcium binding domain.

Animals↗

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

Measurement of two-bond JCOH alpha coupling constants in proteins uniformly enriched with 13C.

A simple E.COSY type technique is described for measurement of two-bond JCOH alpha coupling constants in proteins that are uniformly enriched with 13C. The method has been used to measure 2JCOH alpha for 132 residues in the proteins calmodulin and staphylococcal nuclease having non-overlapping H alpha-C alpha correlations. Measured 2JCOH alpha coupling constants fall in the 0 to -9.5 Hz range. A separate experiment, measuring the accuracy of these values, indicates a root-mean-square error of 1 Hz. Comparison of the J couplings with the dihedral backbone angles from crystallographic studies confirms a weak but statistically significant correlation between the dihedral angle psi and the magnitude of 2JCOH alpha, but also indicates that parameters other than psi have a significant effect on the value of the coupling.

Calmodulin↗

PubMed1992

Quantitative measurement of small through-hydrogen-bond and 'through-space' 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin from Pyrococcus furiosus.

A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin (M(r) approximately 5.4 kDa) from Pyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a 'through-space' coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to 199Hg are significantly larger and fall in the 0.4-4 Hz range.

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↗

PubMed1991

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy.

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.

Alanine↗

PubMed1991

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix.

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.

Amino Acid Sequence↗

PubMed1991

Characterization by NMR of the heme-myoglobin adduct formed during the reductive metabolism of BrCCl3. Covalent bonding of the proximal histidine to the ring I vinyl group.

The reductive debromination of BrCCl3 by ferrous deoxymyoglobin leads to the covalent bonding of the prosthetic heme to the protein. We have previously shown, by the use of peptide mapping and mass spectrometry, that histidine residue 93 is covalently bound to the heme moiety. In the present study the structure of the heme adduct was more completely determined by 1H and 13C NMR techniques. We have found that the ring I vinyl group of the prosthetic heme was altered by the addition of a histidine imidazole nitrogen to the alpha-carbon and a CCl2 moiety to the beta-carbon. The electronic absorption spectra of the oxidized and reduced states of the altered heme-protein indicated that the heme-iron exists in a bis-histidine-ligated form. Analysis of the crystal structure of native myoglobin suggested that for the altered heme-protein, histidine residues 97 and 64 are ligated to the heme-iron and that residue 97 has replaced the native proximal histidine residue 93. These movements, in effect a "histidine shuffle" at the active site, may be responsible for the enhanced reducing activity of the altered protein.

Heme↗

PubMed1991

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta.

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗

PubMed1991

Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination.

A procedure is described that affords complete 1H, 13C and 15N resonance assignment in proteins of up to about 25 kDa. The new approach requires uniform isotopic enrichment of the protein with 13C and 15N and correlates resonances of adjacent nuclei using the relatively large and well-resolved one-bond J couplings. Spectral overlap, a common problem in the application of two-dimensional NMR, is removed by increasing the dimensionality of the new methods to three or four, without increasing the number of observed resonances. With complete 1H, 13C and 15N resonance assignments available, the nuclear Overhauser effect (NOE)-based interproton distance constraints can be extracted in a very straightforward manner from four-dimensional NOE spectra.

Carbon Isotopes↗

PubMed1991

Stereospecific assignment of beta-methylene protons in larger proteins using 3D 15N-separated Hartmann-Hahn and 13C-separated rotating frame Overhauser spectroscopy.

3J alpha beta coupling constants and complementary nuclear Overhauser data on the intraresidue C alpha H-C beta H distances form an essential part of the data needed to obtain stereospecific assignments of beta-methylene protons in proteins. In this paper we show that information regarding the magnitude of the 3J alpha beta coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D 15N-separated 1H-1H Hartmann-Hahn 1H-15N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C alpha H-C beta H distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D 13C-separated 1H-1H rotating frame Overhauser effect 1H-13C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1 beta, a protein of 153 residues and 17.4 kDa molecular weight.

Amino Acids↗

PubMed1991

An efficient 3D NMR technique for correlating the proton and 15N backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15N/13C enriched proteins.

A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C alpha chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from 15N to 13C alpha via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.

Amides↗