Search PubMed⌕ Search

Biomedical subjects

A Bax

Publications and source records attributed to A Bax.

At least 55 records · Page 3Linked to original sources
PubMed1997

Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium.

In isotropic solution, internuclear dipolar couplings average to zero as a result of rotational diffusion. By dissolving macromolecules in a dilute aqueous nematic discotic liquid-crystalline medium containing widely spaced magnetically oriented particles, a tunable degree of solute alignment with the magnetic field can be created while retaining the high resolution and sensitivity of the regular isotropic nuclear magnetic resonance (NMR) spectrum. Dipolar couplings between 1H-1H, 1H-13C, 1H-15N, and 13C-13C pairs in such an oriented macromolecule no longer average to zero, and are readily measured. Distances and angles derived from dipolar couplings in human ubiquitin are in excellent agreement with its crystal structure. The approach promises to improve the accuracy of structures determined by NMR, and extend the size limit.

Crystallization↗
PubMed1997

Refined solution structure and backbone dynamics of HIV-1 Nef.

The tendency of HIV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three-dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel beta-sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non-mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent-exposed loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub-nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determined based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-coupling and ROE data.

Amides↗
PubMed1997

Solution NMR characterization of hydrogen bonds in a protein by indirect measurement of deuterium quadrupole couplings.

Hydrogen bonds stabilize protein and nucleic acid structure, but little direct spectroscopic data have been available for characterizing these critical interactions in biological macromolecules. It is demonstrated that the electric field gradient at the nucleus of an amide hydrogen can be determined residue-specific by measurement of 15N NMR relaxation times in proteins dissolved in D2O, and uniformly enriched with 13C and 15N. In D2O, all backbone amide protons can be exchanged with solvent deuterons, and the T1 relaxation rate of a deuteron is dominated by its quadrupole coupling constant (QCC), which is directly proportional to the electric field gradient at the nucleus. 2HN T1 relaxation can be measured quantitatively through its effect on the T2 relaxation of its directly attached 15N. QCC values calculated from 2HN T1 and previously reported spectral densities correlate with the inverse cube of the X-ray crystal structure-derived hydrogen bond lengths: QCC = 228 + Sigmai 130 cos alphai/ri3 kHz, where alpha is the N-H...Oi angle and r is the backbone-backbone (N-)H...Oi(=C) hydrogen bond distance in ângstroms.

Deuterium↗
PubMed1997

High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid crystalline medium.

A mixture of dihexanoyl phosphatidylcholine and dimyristoyl phosphatidylcholine in water forms disc-shaped particles, often referred to as bicelles [Sanders and Schwonek (1992) Biochemistry, 31, 8898-8905]. These adopt an ordered, liquid crystalline phase, which can be maintained at very low concentrations of the bicelles (down to 3% w/v). At this concentration the spacing between individual bicelles, on average, exceeds 300 A. The bicelles are shown to have a negligible effect on the rotational diffusion of ubiquitin as judged by the 15N T1p values of the backbone amides relative to those in isotropic aqueous solution. The protein exhibits a residual degree of alignment which is proportional to the bicelle concentration, and approximately collinear with ubiquitin's rotational diffusion tensor. The degree of alignment obtained offers unique opportunities for studying the protein's structure and dynamics.

Crystallography↗
PubMed1997

A three-dimensional NMR experiment with improved sensitivity for carbonyl-carbonyl J correlation in proteins.

Recently, a quantitative J correlation technique has been presented that permits measurement of 3Jc'c' in proteins isotopically enriched with 13C [Hu, J.-S. and Bax, A. (1996) J. Am. Chem. Soc., 118, 8170-8171]. Here, we describe an analogous experiment that is less sensitive to transverse 13C' relaxation, which is the principal limiting factor in all 13C-13C long-range correlation experiments on macromolecules. The new scheme utilizes homonuclear Hartmann-Hahn cross polarization (TOCSY) instead of a COSY type transfer to accomplish magnetization transfer; a description of the relevant relaxation terms is presented. The experiment is demonstrated for ubiquitin and HIV-1 Nef. The results show excellent agreement between 3Jc'c' values measured for ubiquitin with the new scheme and those reported previously. The experiment is particularly useful for distinguishing backbone phi angles that are smaller than -120 degrees from those larger than -120 degrees.

Amino Acid Sequence↗
PubMed1997

Chi 1 angle information from a simple two-dimensional NMR experiment that identifies trans 3JNC gamma couplings in isotopically enriched proteins.

New quantitative J correlation experiments are used for measuring all two- and three-bond couplings between 15N and aliphatic side-chain carbons in proteins uniformly enriched in 13C and 15N. Results show that 3JNC beta and 2JNC beta invariably are very small. Therefore, a simple and relatively sensitive two-dimensional spin-echo difference experiment can be used to identify residues with a 3JN gamma coupling substantially larger than 1 Hz, indicative of a trans arrangement between N and C gamma. This measurement therefore provides chi 1 angle information for residues with an aliphatic C gamma carbon, and thereby also aids in making stereospecific assignments of H beta resonances. Experiments are demonstrated for ubiquitin and for a complex between calmodulin and a 26-residue peptide.

Amino Acid Sequence↗
PubMed1997

Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy.

Structure determination by NMR presently relies on short range restraints between atoms in close spatial proximity, principally in the form of short (< 5 A) interproton distances. In the case of modular or multidomain proteins and linear nucleic acids, the density of short interproton distance contacts between structural elements far apart in the sequence may be insufficient to define their relative orientations. In this paper we show how the dependence of heteronuclear longitudinal and transverse relaxation times on the rotational diffusion anisotropy of non-spherical molecules can be readily used to directly provide restraints for simulated annealing structure refinement that characterize long range order a priori. The method is demonstrated using the N-terminal domain of Enzyme I,a protein of 259 residues comprising two distinct domains with a diffusion anisotropy(Dparallel/Dperpendicular)of approximately 2.

Anisotropy↗
PubMed1997

Use of dipolar 1H-15N and 1H-13C couplings in the structure determination of magnetically oriented macromolecules in solution.

Anisotropy of the molecular magnetic susceptibility gives rise to a small degree of alignment. The resulting residual dipolar couplings, which can now be measured with the advent of higher magnetic fields in NMR, contain information on the orientation of the internuclear vectors relative to the molecular magnetic susceptibility tensor, thereby providing information on long range order that is not accessible by any of the solution NMR parameters currently used in structure determination. Thus, the dipolar couplings constitute unique and powerful restraints in determining the structures of magnetically oriented macromolecules in solution. The method is demonstrated on a complex of the DNA-binding domain of the transcription factor GATA-1 with a 16 base pair oligodeoxyribonucleotide.

Anisotropy↗
PubMed1996

A T cell receptor V alpha domain expressed in bacteria: does it dimerize in solution?

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR V alpha from a hybridoma with specificity for the human immunodeficiency virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2Dd. This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-V alpha 2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of beta sheet. Conditions for crystallization were established, and at least two crystal geometries were observed: hexagonal bipyramids that failed to diffract beyond approximately 6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the V alpha domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at a concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some V alpha domains have little tendency to form homodimers or multimers.

Amino Acid Sequence↗
PubMed1996

The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR.

Using heteronuclear NMR spectroscopy, we demonstrate that a 13-residue peptide (MS-QIKRLLSEKKT) from the cytoplasmic tail of CD4 binds to Nef protein. This part of CD4 is critical for downregulation of CD4 by HIV-1 Nef [Aiken et al. (1994) Cell 76, 853-864]. We show that a control peptide without the central dileucine does not bind to Nef. The dependence of Nef 1H and 15N amide chemical shifts on peptide concentration indicates that the binding is in the fast chemical exchange limit, with a dissociation constant Kd of approximately 1 mM. The peptide binding site has been mapped onto the previously determined solution structure of HIV-1 Nef [Grzesiek et al. (1996) Nat. Struct. Biol. 3, 340-345] on the basis of peptide-induced chemical shift changes. It comprises amino acids W57, L58, E59, G95, G96, L97, R106, and L110. When Nef is complexed to the SH3 domain of Hck tyrosine protein kinase, the peptide binds to the same site on Nef but with slightly higher affinity (Kd approximately 0.5 mM). This indicates that the binding of CD4 and Hck SH3 to Nef are two compatible and slightly cooperative events.

Amides↗
PubMed1996

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields.

15N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz 1H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T1/T2 ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the 15N T1 and T2 relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T1 values measured at 360 and 600 MHz is 0.50 +/- 0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with tau c = 10.7 ns. The average ratio of the T2 values measured at 360 and 600 MHz is 1.14 +/- 0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T1 and T2 relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in 15N relaxation studies of proteins.

Anisotropy↗
PubMed1996

The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase.

The solution structure of HIV-1 Nef has been solved by multidimensional heteronuclear NMR spectroscopy. The construct employed to circumvent problems associated with aggregation was a double-deletion mutant (delta2-39, delta159-173) in which conformationally disordered regions of the protein at the N terminus and in a long solvent-exposed flexible loop were removed, without affecting the properties or structural integrity of the remainder of the protein. Despite the absence of any sequence similarity, the overall fold of Nef is reminiscent of that of the family of winged helix-turn-helix DNA binding proteins. The binding surface of Nef for the SH3 domain of Hck tyrosine protein kinase has been mapped and reveals a non-contiguous (in terms of amino-acid sequence) interaction surface. This unique feature may suggest possible avenues for drug design aimed at inhibiting the interaction between Nef and SH3 domains.

Amino Acid Sequence↗
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↗