The Devi case and more.

Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal-coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c3. NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (nu4, nu3, and nu2) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c3 (Norway 4 and 9974) than for the Desulfovibrio (D.) gigas, D. vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the nu3 and nu10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins.

Crystallography, X-Ray↗

PubMed1998

NMR solution structure of the N3' --> P5' phosphoramidate duplex d(CGCGAATTCGCG)2 by the iterative relaxation matrix approach.

High-resolution 2D NMR spectra of the duplex CGCGAATTCGCG with deoxyribose sugars but with the normal phosphodiester linker replaced by an N3' --> P5' phosphoramidate (NP) group have been used to establish a solution structure for the duplex. Distance, angle, and base pair hydrogen-bonding constraints were used to refine the structure by use of the iterative relaxation matrix approach (IRMA). The phosphoramidate NH proton signal could be observed in DMSO at low temperature but not in H2O and D2O. For this reason, the structure was refined with the -NH in each of the two possible low-energy configurations. The structure with the nitrogen lone pair located between the nonbridging oxygen atoms of the 5'-phosphate group consistently had the lowest energy and RMSD values, consistent with an X-ray analysis of the same duplex [Tereshko, V., Gryaznov, S. , and Egli, M. (1998) J. Am. Chem. Soc. 120, 269-283]. In the refined structure, the sugars are in the C3'-endo conformation with the change from the normal C2'-endo conformation of deoxyribose apparently being driven by the gauche effect and the change in electronegativity from the 3'O to the 3'NH group. In agreement with preliminary studies [Ding, D., Gryaznov, S. M., Lloyd, D. H., Chandrasekaran, S., Yao, S., Ratmeyer, L., Pan, Y., and Wilson, W. D. (1996) Nucleic Acids Res. 24, 354-360], the backbone conformation in the NP duplex is very close to classical A-form values. Comparison of phosphodiester and phosphoramidate structures suggests that their backbones have global conformations that are primarily a function of the low-energy state of the sugar ring. A somewhat more complex situation arises when base pair conformation is analyzed with many of the base pairs having a conformation between those of classical A- and B-form helices. The effects of the 2' substituent are obviously important in specifying the final conformation of the stacked bases in either an A-form or B-form helix. It is clear, however, that conversion of the normal phosphodiester of DNA into a phosphoramidate linkage yields a nucleic acid that behaves much more like RNA than DNA, and it has been shown that NP sequences can bind to RNA-directed proteins [Rigl, C. T., Lloyd, D. H., Tsou, D. S., Gryaznov, S. M., and Wilson, W. D. (1997) Biochemistry 36, 650-659].

Crystallography, X-Ray↗