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V J Basus

Publications and source records attributed to V J Basus.

10 recordsLinked to original sources
PubMed1992

Sequence-specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained.

Animals↗
PubMed1991

Protein solution structure determination using distances from two-dimensional nuclear Overhauser effect experiments: effect of approximations on the accuracy of derived structures.

Solution structures for many proteins have been determined to date utilizing interproton distance constraints estimated from two-dimensional nuclear Overhauser effect (2D NOE) spectra. Although the simple isolated spin pair approximation (ISPA) generally used can result in systematic errors in distances, the large number of constraints enables protein structure to be defined with reasonably high resolution. Effects of these systematic errors on the resulting protein structure are examined. Iterative relaxation matrix calculations, which account for dipolar interactions between all protons in a molecule, can accurately determine internuclear distances with little or no a priori knowledge of the molecular structure. The value of this additional complexity is also addressed. To assess these distance determination methods, hypothetical "experimental" data, including random noise and peak overlap, are calculated for an arbitrary "true" protein structure. Three methods of obtaining distance constraints from 2D NOE peak intensities are examined: one entails a conservative use of ISPA, one assumes the ISPA to be fairly accurate, and one utilizes an iterative relaxation matrix method called MARDIGRAS (matrix analysis of relaxation for discerning the geometry of an aqueous structure), developed in this laboratory. A distance geometry algorithm was used to generate a family of structures for each distance set. The quality of the average structure from each family was good. The root-mean-square deviation of that average structure from the true structure was improved about 2-5% using the more restrictive rather than the more conservative ISPA approach. Use of MARDIGRAS in a conservative fashion--i.e., with a poor initial model--resulted in improvement in the root-mean-square deviation by 8-15%. With a better initial model, MARDIGRAS obtained even more accurate distances. MARDIGRAS also permits analysis of 2D NOE data at longer mixing times, yielding additional distances. Use of more restrictive ISPA distances did, however, result in a few systematically incorrect structural features in local regions of the protein, producing distortions of 2-3 A. Comparison between experimental data and spectra calculated for the structures correlates with root-mean-square deviation, offering a method of structure evaluation. An R factor for evaluating fit between experimental and calculated 2D NOE intensities is proposed.

Magnetic Resonance Spectroscopy↗
PubMed1989

Proton nuclear magnetic resonance assignments.

The procedures outlined here have been used successfully for more than 30 proteins to date, and are nearly routine for molecules up to a molecular weight of 10,000. Some of the proteins assigned have a molecular weight greater than 10,000. For these larger proteins, relayed-COSY and TOCSY experiments have been essential for the identification of spin systems, although for thioredoxin these experiments could not be used. In this case, assignments were accomplished using nonspecific deuteration to the level of 75% and specific, nearly complete, deuteration of certain kinds of residues (see LeMaster [2], this volume). Nonspecific deuteration reduces the cross-relaxation rates of each proton to the rest of the molecule, thus reducing the linewidths. The cross-peak patterns were also narrowed due to simplification of the coupling patterns. Such a laborious procedure of nonspecific deuteration may not be necessary for complete proton assignments of proteins in this size range, as evidenced by the fact that this method was not used for the other two molecules mentioned above. It may prove, however, to be quite valuable in the study of larger molecules, where linewidths are expected to increase due to longer rotational correlation times. Overlap problems in the NH chemical shifts can be dealt with by making use of the differential temperature dependence of these shifts. Another technique is to take advantage of the wide range of exchange rates between these protons and the solvent. Spectra containing only the slowly exchanging NH protons can be obtained by acquiring spectra of the protein soon after dilution in D2O, and spectra of only the rapidly exchanging protons can be obtained by obtaining spectra in a freshly prepared H2O solution of the protein after having completely exchanged all the NH protons with deuterium. Variation of the pH will resolve problems of overlap in all regions of the spectrum, although many chemical shifts may be unaffected by pH. In some cases, pH variation may change the conformation of the molecule. This may, in fact, assist in the sequential assignment if the chemical shifts can be followed with pH. Finally, the relayed-NOESY experiments can resolve overlap problems with the alpha-proton chemical shifts. Thus, it is very likely that the assignment methods outlined here will be successful for the assignment of the proton spectra of even larger molecules if there is significant secondary structure and significant variety of residues to provide enough dispersion of the chemical shifts.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1988

Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments.

We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)].

Amino Acid Sequence↗
PubMed1988

Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment.

Complete sequence-specific assignments of the 1H NMR spectrum of bungarotoxin were reported in the previous paper [Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., & Kuntz, I.D. (1988) Biochemistry (first paper of three in this issue)]. The assignment was significantly aided by the use of the homonuclear Hartman-Hahn relayed coherence transfer nuclear Overhauser enhancement spectroscopy experiment (HRNOESY) which we present here, as a modification of relayed coherence transfer nuclear Overhauser enhancement spectroscopy (relayed NOESY) [Wagner, G. (1984) J. Magn. Reson. 57, 497]. As shown here, HRNOESY resolves problems of proton resonance overlap especially in extended chain conformations as found in beta-sheets.

Bungarotoxins↗
PubMed1988

Structural studies of alpha-bungarotoxin. 3. Corrections in the primary sequence and X-ray structure and characterization of an isotoxic alpha-bungarotoxin.

The most plausible set of chemical shift assignments for alpha-bungarotoxin as deduced from the combined use of two-dimensional J-correlated and two-dimensional nuclear Overhauser effect 1H nuclear magnetic resonance (NMR) spectroscopy was in conflict with the accepted amino acid sequence between residues 8 and 12 and residues 66 and 70 [Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M., & Kuntz, I. D. (1988) Biochemistry (first paper of three in this issue]). Furthermore, NMR spectra of alpha-bungarotoxin, purified by conventional methods, evidenced a second species at the level of approximately 10% total protein. The minor component was separated from alpha-bungarotoxin by Mono-S (cationic) chromatography. Sequencing of Mono-S-purified alpha-bungarotoxin and one of its tryptic peptides showed that the correct sequence for alpha-bungarotoxin is Ser-Pro-Ile at positions 9-11 and Pro-His-Pro at positions 67-69. The electron density map of alpha-bungarotoxin [Love, R. A., & Stroud, R. M. (1986) Protein Eng. 1, 37] was refined with the new sequence data. Improvements in the structure were found primarily for residues 9-11. Sequence analysis of two overlapping tryptic peptides proved that the minor species differed from alpha-bungarotoxin by replacement of a valine for an alanine at position 31. This new toxin, alpha-bungarotoxin(Val-31), binds to the acetylcholine receptor with an affinity that is comparable to that of alpha-bungarotoxin.

Amino Acid Sequence↗
PubMed1986

Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor.

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.

Amino Acids↗
PubMed1985

Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission.

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.

Bleomycin↗