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A Bax

Publications and source records attributed to A Bax.

At least 19 recordsLinked to original sources
PubMed1995

NMR identification of calcineurin B residues affected by binding of a calcineurin A peptide.

Triple resonance 3D NMR methods have been used to study the interaction between calcineurin B and a peptide fragment of calcineurin A for which it has high affinity (KD approximately 4 x 10(-7) M). Although calcineurin B aggregates at NMR concentrations of approximately 1 mM, in the presence of a target peptide fragment of calcineurin A it becomes monomeric and yields NMR spectra that are very similar to those reported previously for calcineurin B solubilized by the zwitterionic detergent CHAPS. Changes in chemical shifts between CHAPS- and peptide-solubilized calcineurin B are small which is indicative of no differences in secondary structure. Residues most affected by binding to target peptide are found primarily on the hydrophobic faces of the four helices, present in each of the two globular domains in calcineurin B, and in the loops connecting helices II and III, IV and V, and possibly in the C-terminal 12 residues, which also exhibit a change in mobility.

Amino Acid Sequence↗
PubMed1995

Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements.

The backbone motions of calcium-free Xenopus calmodulin have been characterized by measurements of the 15N longitudinal relaxation times (T1) at 51 and 61 MHz, and by conducting transverse relaxation (T2), spin-locked transverse relaxation (T1 rho), and 15N-[1H] heteronuclear NOE measurements at 61 MHz 15N frequency. Although backbone amide hydrogen exchange experiments indicate that the N-terminal domain is more stable than calmodulin's C-terminal half, slowly exchanging backbone amide protons are found in all eight alpha-helices and in three of the four short beta-strands. This confirms that the calcium-free form consists of stable secondary structure and does not adopt a 'molten globule' type of structure. However, the C-terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 microseconds, which affects many of the C-terminal domain residues. This results in significant shortening of the 15N T2 values relative to T1 rho, whereas the T1 rho and T2 values are of similar magnitude in the N-terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the T1 values and does not explain the different T2 values found for different alpha-helices in the N-terminal domain. These latter parameters are compatible with a flexible dumb-bell model in which each of calmodulin's two domains freely diffuse in a cone with a semi-angle of about 30 degrees and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1-17].

Animals↗
PubMed1995

Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N.

It is demonstrated that sequential resonance assignment of the backbone 1H alpha and 15N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate 1H alpha with the intraresidue 15N and with the 15N resonance of the next residue. The experiments are preferably conducted in D2O, where very high resolution in the 15N dimension can be achieved by using 2H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in 13C and 15N. Complete backbone and 13C beta/1H beta resonance assignments are presented.

Amino Acid Sequence↗
PubMed1995

Flexibility and function in HIV-1 protease.

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the microsecond time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

Amino Acid Sequence↗
PubMed1995

Solution structure of calcium-free calmodulin.

The three-dimensional structure of calmodulin in the absence of Ca2+ has been determined by three- and four-dimensional heteronuclear NMR experiments, including ROE, isotope-filtering combined with reverse labelling, and measurement of more than 700 three-bond J-couplings. In analogy with the Ca(2+)-ligated state of this protein, it consists of two small globular domains separated by a flexible linker, with no stable, direct contacts between the two domains. In the absence of Ca2+, the four helices in each of the two globular domains form a highly twisted bundle, capped by a short anti-parallel beta-sheet. This arrangement is qualitatively similar to that observed in the crystal structure of the Ca(2+)-free N-terminal domain of troponin C.

Animals↗
PubMed1994

1H, 13C, 15N nuclear magnetic resonance backbone assignments and secondary structure of human calcineurin B.

The calmodulin- and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with 15N and 13C. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.

Amino Acid Sequence↗
PubMed1994

Localization of bound water in the solution structure of a complex of the erythroid transcription factor GATA-1 with DNA.

BACKGROUND: The erythroid specific transcription factor GATA-1 is responsible for the regulation of transcription of erythroid-expressed genes and is an essential component required for the generation of the erythroid lineage. GATA-1 binds specifically as a monomer to the asymmetric consensus target sequence (T/A)GATA-(A/G) found in the cis-regulatory elements of all globin genes and most other erythroid specific genes that have been examined. We have previously determined the solution structure of the complex of the zinc-containing DNA-binding domain of chicken GATA-1 with its cognate DNA target site by multidimensional heteronuclear NMR. From previous studies of complexes between proteins and DNA, water appears to play an important role in DNA-protein recognition by mediating bridging hydrogen bonds between functional groups on the protein and DNA bases. Solvation free energy calculations, however, suggest that hydrophobic interactions should exclude water from parts of the GATA-1:DNA interface. RESULTS: Using water-selective two-dimensional heteronuclear magnetic resonance spectroscopy, we have identified the location of bound water molecules in the specific complex of chicken GATA-1 with DNA. A number of water molecules could be detected between the protein and the phosphate backbone, as well as at the solvent exposed surface of the protein. However, no water molecules could be observed at the interface of the protein with the bases of the DNA. With only one exception, the bound water molecules have a residency time > 200-300 ps. CONCLUSIONS: Unlike other protein-DNA complexes, the majority of specific interactions between GATA-1 and the DNA bases in the major groove are hydrophobic in nature. The exclusion of water from the protein-DNA base interface in the major groove supports the view that the specific binding energy is indeed dominated by hydrophobic effects.

Animals↗
PubMed1994

NMR evidence for similarities between the DNA-binding regions of Drosophila melanogaster heat shock factor and the helix-turn-helix and HNF-3/forkhead families of transcription factors.

Heteronuclear multidimensional NMR experiments of residues 33-163 of the DNA-binding domain of Drosophila heat shock factor, dHSF(33-163), were recorded, using only 3 mg of uniformly 15N-labeled or 2 mg of uniformly 15N/13C-labeled protein. The polypeptide consists of a structured part comprising three helices, a three-stranded antiparallel beta-sheet, with the first two strands connected by a four-residue type I tight turn. The second helix is disrupted at its C-terminal end by a proline residue and is followed by an extended turn, leading to the third helix. The dHSF(33-163) protein is unstructured at its N- and C-termini, and a third unstructured region is found from Thr113 to Arg124. Exchange broadening of the 15N-1H correlations upon titration of 15N labeled HSF with a 13-base-pair DNA duplex suggests a DNA-binding motif in which the third helix acts as the recognition helix. Both the secondary structure and DNA-binding pattern of dHSF(33-163) suggest that the overall topology resembles that the helix-turn-helix bacterial activator CAP [Weber, I. T., & Steitz, T. A. (1987) J. Mol. Biol. 198, 311-326] and the liver-specific transcription factor HNF-3 gamma, the prototype of the HNF-3/forkhead protein family [Clark, K. L., Halay, E. D., Lai, E., & Burley, S. K. (1993) Nature 364, 412-420].

Amino Acid Sequence↗
PubMed1994

Measurement of four-bond HN-H alpha J-couplings in staphylococcal nuclease.

Quantitative J-correlation and triple-resonance ECOSY-type experiments are used to unambiguously establish the presence of four-bond sequential HN-H alpha J-couplings in the protein staphylococcal nuclease. Substantially negative 4JH alpha HN values, ranging from -0.8 to -2.3 Hz, are observed when the psi angle is near +120 degrees, and the following phi angle near +60 degrees. For other conformations, the four-bond HN-H alpha J-couplings fall between -0.5 and +0.5 Hz.

Amino Acid Sequence↗
PubMed1994

Resonance assignment of methionine methyl groups and chi 3 angular information from long-range proton-carbon and carbon-carbon J correlation in a calmodulin-peptide complex.

Several simple 3D experiments are used to provide J correlations between methionine C epsilon methyl carbons and either the C gamma H2 protons or C beta and C gamma. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the chi 3 torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the C epsilon H3 methyl protons and C gamma. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.

Amino Acid Sequence↗
PubMed1994

Measurement of HN-H alpha J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods.

Two new methods are described for the measurement of three-bond JHNH alpha couplings in proteins isotopically enriched with 15N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNH alpha, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80 have JHNH alpha values in the 6-7 Hz range, suggesting that a break in the 'central helix' occurs at the same position as previously observed in solution NMR studies of Ca(2+)-ligated calmodulin.

Calmodulin↗
PubMed1994

Solution structure of the DNA-binding domain of Drosophila heat shock transcription factor.

The solution structure of the DNA-binding domain of the Drosophila heat shock transcription factor, as determined by multidimensional multinuclear NMR, resembles that of the helix-turn-helix class of DNA-binding proteins. The domain comprises a four-stranded antiparallel beta-sheet, packed against a three-helix bundle. The second helix is significantly distorted and is separated from the third helix by an extended turn which is subject to conformational averaging on an intermediate time scale. Helix 3 forms a classical amphipathic helix with polar and charged residues exposed to the solvent. Upon titration with DNA, resonance shifts in the backbone and Asn and Gln side-chain amides indicate that helix 3 acts as the recognition helix of the heat shock transcription factor.

Amino Acid Sequence↗
PubMed1993

A simple and sensitive experiment for measurement of JCC couplings between backbone carbonyl and methyl carbons in isotopically enriched proteins.

A simple 2D difference experiment is described that allows quantitative measurement of 13C-13C J couplings between backbone carbonyl and side-chain carbons. Precise 3JCC values were measured from data recorded in just 2 h for a 1-mM solution of the 20-kD complex between the protein calmodulin and a 26-residue synthetic peptide. The J couplings aid in determining the chi 1 angles of valine, isoleucine and threonine residues, and in making stereospecific assignments of the Val C gamma methyl groups. Error analysis indicates that the uncertainty in the derived J couplings is generally less than ca. 0.3 Hz.

Amino Acids↗
PubMed1993

Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B.

A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly 13C/15N-enriched proteins and can measure magnetization exchange rates in the 0.02 to > 20 s-1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H alpha resonances, followed by NOESY- or ROESY-type transfer to amide protons, which are dispersed by the amide 15N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H2O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the 'central helix' of the homologous protein calmodulin.

Amides↗
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↗