The backbone dynamics of the major coat protein (gVIIIp) of the filamentous bacteriophage M13, solubilized in detergent micelles, have been studied using 15N nuclear magnetic resonance spectroscopy at three frequencies. Motional parameters and overall and internal correlation times were derived with the model-free approach. It was also checked whether these parameters had to be modified due to anisotropic motion of the protein/micelle complex. Reduced spectral density mapping was used to calculate the spectral densities at J(O), J(omegaN), and [J(omegaH)]. The spectral densities were interpreted by mapping a linear or scaled linear combination of two Lorentzians onto a J(O)-J(omega) plot. The major coat protein of bacteriophage M13 consists of two alpha-helices, one of which is hydrophobic and located within the micelle, while the other is amphipathic and located on the surface of the micelle. Our results indicate that the motion of the hydrophobic helix is restricted such that it corresponds to the overall tumbling of the protein/micelle complex. The interpretation of the relaxation data of the amphipathic helix by means of the model-free approach and the reduced spectral density mapping indicate that in addition to the overall motion all residues in this helix are subject to motion on the fast nanosecond and picosecond time scales. The motions of the vectors in the low nanosecond range are characterized by similar values of the spectral densities and correlation times and represent the motion of the amphipathic helix on and away from the surface of the micelle. The relaxation data of the residues in the hinge region connecting the helices show that there is an abrupt change from highly restricted to less restricted motion. Both the C-terminal and N-terminal residues are very mobile.

Sequential assignments and secondary structural analysis have been accomplished for the 113-residue apoprotein of the antitumor drug neocarzinostatin (NCS) from Streptomyces carzinostaticus. A total of 98% of the main-chain and 77% of the side-chain resonances have been sequence specifically assigned by use of information from coherence transfer experiments and by sequential and interstrand NOEs. Because of the complexity of the NCS spectrum, several sequential assignment strategies were employed to complete the analysis. Apo-NCS consists of three antiparallel beta-sheeted domains by NMR analysis. There is an extensive four-strand antiparallel beta-sheet, and two two-stranded domains. One of the two-strand domains is contiguous, S72-N87, with chain reversal occurring through the region L77-R82. The other two-stranded domain has the section G16-A24 antiparallel with respect to the region S62-R70. This secondary structure is consistent with the crystal structure of holo-NCS at 2.8-A resolution.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pK, of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 'HN, I5N, I3Ca, "CO, 'Ha, and I3Cp NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple ( 'H-'3C-'5N) and double resonance ('H-'5N or 'H-I3C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, ? T H~H U and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro15 1, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large I3Ca chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.

1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)-CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the alpha-protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H alpha chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020-9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C alpha and H alpha frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C alpha chemical shifts and 'TOCSY ladders'. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C alpha secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427-445] shows no major differences.

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