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A Bax

Publications and source records attributed to A Bax.

At least 37 records · Page 2Linked to original sources
PubMed2000

Study of conformational rearrangement and refinement of structural homology models by the use of heteronuclear dipolar couplings.

For an increasing fraction of proteins whose structures are being studied, sequence homology to known structures permits building of low resolution structural models. It is demonstrated that dipolar couplings, measured in a liquid crystalline medium, not only can validate such structural models, but also refine them. Here, experimental 1H-15N, 1Halpha-13Calpha, and 13C'-13Calpha dipolar couplings are shown to decrease the backbone rmsd between various homology models of calmodulin (CaM) and its crystal structure. Starting from a model of the Ca2+-saturated C-terminal domain of CaM, built from the structure of Ca2+-free recoverin on the basis of remote sequence homology, dipolar couplings are used to decrease the rmsd between the model and the crystal structure from 5.0 to 1.25 A. A better starting model, built from the crystal structure of Ca2+-saturated parvalbumin, decreases in rmsd from 1.25 to 0.93 A. Similarly, starting from the structure of the Ca2+-ligated CaM N-terminal domain, experimental dipolar couplings measured for the Ca2+-free form decrease the backbone rmsd relative to the refined solution structure of apo-CaM from 4.2 to 1.0 A.

Amino Acid Sequence↗
PubMed2000

Solution structure of DinI provides insight into its mode of RecA inactivation.

The Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81-residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an alpha/beta fold comprised of a three-stranded beta-sheet and two alpha-helices. The beta-sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C-terminal alpha-helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single-stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.

Aspartic Acid↗
PubMed1999

Solution structure of anti-HIV-1 and anti-tumor protein MAP30: structural insights into its multiple functions.

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30's apparent inhibition of the HIV-1 integrase, MAP30's ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30's anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30's glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

Anti-HIV Agents↗
PubMed1999

Observation of through-hydrogen-bond 2hJHC' in a perdeuterated protein.

It is demonstrated that J connectivity between amide protons and hydrogen-bond-accepting carbonyl carbons can be observed in perdeuterated human ubiquitin. A selective pulse scheme is used to detect these small 2hJHC' interactions in the presence of the much larger through-covalent-bond 2JHC' and 3JHC' couplings. The ratio of the observed through-H-bond correlation intensity and the 2JHC' connectivity observed in a reference spectrum indicates 2hJHC' values of ca. 0.4-0.6 Hz, which are only slightly smaller than the corresponding 3hJNC' values. However, for technical reasons, 2hJHC' couplings are more difficult to measure than 3hJNC'.

Amides↗
PubMed1999

Measurement of 3hJNC' connectivities across hydrogen bonds in a 30 kDa protein.

A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons.

Carbon Isotopes↗
PubMed1999

Protein backbone angle restraints from searching a database for chemical shift and sequence homology.

Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C alpha, 13C beta, 13C', 1H alpha and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar phi and psi backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 degrees. Approximately 3% of the predictions made by TALOS are found to be in error.

Amino Acid Sequence↗
PubMed1999

Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values.

It is demonstrated that mixtures of ditetradecyl-phosphatidylcholine or didodecyl-phoshatidylcholine and dihexyl-phosphatidylcholine in water from lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). The carboxy-ester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid- or base-catalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3-10.4 pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range.

Chemical Phenomena↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Databases as Topic↗
PubMed1998

Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra.

Simple procedures are described for recording complementary in-phase and antiphase J-coupled NMR spectra. The sum and difference of these spectra contain only the upfield and the downfield components of a doublet, making it possible to measure the J splitting directly from these combinations without an increase in resonance overlap relative to the decoupled spectrum. The approach is demonstrated for measurement of 1JNH splittings and 2JHNC splittings in oriented and isotropic ubiquitin. Dipolar couplings obtained from differences in the splittings measured in the oriented and isotropic phases are in excellent agreement with dipolar couplings obtained from direct measurement of the splitting or from a conventional E. COSY-type measurement.

Image Processing, Computer-Assisted↗
PubMed1998

A robust method for determining the magnitude of the fully asymmetric alignment tensor of oriented macromolecules in the absence of structural information.

It has recently been shown that the degree of alignment of macromolecules in an aqueous dilute liquid crystalline medium of bicelles is sufficient to permit accurate values of residual 15N-1H, 13C-1H, and 13Calpha-C' dipolar couplings to be obtained on a routine basis, thereby providing potentially unique long-range structural information. To make use of this information in macromolecular structure determination, the magnitude of the axial and rhombic components of the molecular alignment tensor must be determined. This can be achieved by taking advantage of the fact that different, fixed-distance internuclear vector types are differently distributed relative to the alignment tensor. A histogram of the ensemble of normalized residual dipolar couplings for several such vector types approximates a powder pattern from which the magnitude of the axial and rhombic components are readily extracted in the absence of any prior structural information. The applicability of this method is demonstrated using synthetic data derived from four proteins representative of different sizes, topologies, and secondary structures, and experimental data measured on the small protein ubiquitin.

Carbon↗
PubMed1998

Determination of three-bond 1H3'-31P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy.

A new sensitive two-dimensional quantitative J correlation experiment is described for measuring 3JH3'-P couplings in nucleic acids and protein-nucleic acid complexes. The method is based on measuring the change in intensity of the 1H-1H cross peaks in a constant-time 1H-1H COSY experiment which occurs in the presence and absence of 3JH3'-P dephasing during the constant-time evolution period. For protein-nucleic acid complexes where the protein is 13C-labeled but the nucleic acid is not, 12C-filtering is readily achieved by the application of a series of 13C purge pulses during the constant time evolution period without any loss of signal-to-noise of the nucleic acid cross peaks. The method is demonstrated for the Dickerson DNA dodecamer and a 19 kDa complex of the transcription factor SRY with a 14mer DNA duplex. The same approach should be equally applicable to numerous other problems, including the measurement of JH-Cd couplings in cadmium-ligated proteins, or 3JCH couplings in other selectively enriched compounds.

Base Sequence↗
PubMed1998

Measurement of dipolar couplings for methylene and methyl sites in weakly oriented macromolecules and their use in structure determination.

A simple and effective method is described for simultaneously measuring dipolar couplings for methine, methylene, and methyl groups in weakly oriented macromolecules. The method is a J-modulated 3D version of the well-known [1H-13C] CT-HSQC experiment, from which the J and dipolar information are most accurately extracted by using time-domain fitting in the third, constant-time dimension. For CH2-sites, the method generally yields only the sum of the two individual 13C-1H couplings. Structure calculations are carried out by minimizing the deviation between the measured sum, and the sum predicted for each methylene on the basis of the structure. For rapidly spinning methyl groups the dipolar contribution to the splitting of the outer 13C quartet components can be used directly to constrain the orientation of the C-CH3 bond. Measured sidechain dipolar couplings are in good agreement with an ensemble of NMR structures calculated without use of these couplings.

Humans↗
PubMed1998

Measurement of three-bond, 13C'-13C beta J couplings in human ubiquitin by a triple resonance, E. COSY-type NMR technique.

A [CO]HN(CA)CB-E.COSY pulse scheme is described for measurement of three-bond couplings, 3JC'C beta, between carbonyl and aliphatic C beta carbons in ubiquitin, uniformly enriched with 13C and 15N. A Karplus relation, 3JC'C beta = 1.28 cos2(phi - 120 degrees) -1.02 cos(phi - 120 degrees) + 0.30 Hz, is obtained by correlating the 3JC'C beta values measured for human ubiquitin with backbone phi angles from its crystal structure. As predicted, the new Karplus parametrization yields 3JC'C beta values slightly larger than previously obtained by quantitative J correlation [Hu, J.-S. and Bax, A. (1997) J. Am. Chem. Soc., 119, 6360-6368], but considerably smaller than what has been reported on the basis of other E.COSY-type measurements carried out on flavodoxin.

Carbon Isotopes↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
PubMed1998

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules.

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of 1H, 13C or 15N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 degrees C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.

Crystallization↗
PubMed1998

Solution structure of cyanovirin-N, a potent HIV-inactivating protein.

The solution structure of cyanovirin-N, a potent 11,000 Mr HIV-inactivating protein that binds with high affinity and specificity to the HIV surface envelope protein gp120, has been solved by nuclear magnetic resonance spectroscopy, including extensive use of dipolar couplings which provide a priori long range structural information. Cyanovirin-N is an elongated, largely beta-sheet protein that displays internal two-fold pseudosymmetry. The two sequence repeats (residues 1-50 and 51-101) share 32% sequence identity and superimpose with a backbone atomic root-mean-square difference of 1.3 A. The two repeats, however, do not form separate domains since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Analysis of surface hydrophobic clusters suggests the location of potential binding sites for protein-protein interactions.

Amino Acid Sequence↗