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Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP (1999)

Babini, Elena ; Borsari, Marco ; Capozzi, Francesco ; [et al.]

Springer

JBIC 4 (1999), S. 692-700

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ISSN: 1432-1327

Keywords: Key words Chromatium vinosum ; Electrostatic effects ; High-potential iron-sulfur protein ; NMR spectroscopy ; Reduction potential

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology

Notes: Abstract The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with Sγ of Cys77, one of the cluster ligands. The reduction potential of the S79P variant was 104±3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins. These results suggest that the influence of amide group 79 on the reduction potential of C. vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s007750050341

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Assignment of Pseudo-contact-shifted 1H NMR resonances in the EF site of Yb3 +-substituted rabbit parvalbumin through a combination of 2D techniques and magnetic susceptibility tensor determination (1993)

Capozzi, Francesco ; Cremonini, Mauro Andrea ; Luchinat, Claudio ; [et al.]

Chichester : Wiley-Blackwell

Organic Magnetic Resonance 31 (1993), S. S118

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ISSN: 0749-1581

Keywords: 1H NMR ; Pseudo-contact shift ; Magnetic susceptibility tensor ; Rabbit parvalbumin ; Chemistry ; Analytical Chemistry and Spectroscopy

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Chemistry and Pharmacology

Notes: Pseudo-contact-shifted 1H NMR resonances of rabbit parvalbumin substituted with Yb3+ at the Ca2+ EF site have been sequence-specific assigned by a combination of 2D techniques and magnetic susceptibility tensor determination. By taking the X-ray structure of the homologous carp protein as a starting point, the sequence-specific assignment proceeded in a stepwise fashion. The first resonances assigned on the basis of their unique connectivity pattern were used to make a first coarse determination of the X-tensor parameters; the latter were used to guess the identity of further hyperfine-shifted resonances, which were then checked through 2D connectivities and in turn allowed us to improve the reliability of the X-tensor parameters. This cyclic procedure was repeated until virtually all pseudo-contact-shifted resonances were assigned. In the last steps the X-tensor parameters did not change appreciably. The orientation of the tensor was found to be very similar to that previously proposed for the homologous Yb3+-substituted protein from carp. Modest discrepancies between some calculated and experimental shift values in the present system were found to be due mainly to structural differences between the present protein and the homologous protein whose x-ray structure was used as model and, to a lesser extent, to indetermination in the diamagnetic shift values. The success of the analysis and the univocal solution for the X-tensor parameters under these conditions show that the above procedure is robust enough to permit both signal assignment and X-tensor determination even when the structural data are only a rough approximation of the actual structure. These findings may open the way to the development of a systematic use of pseudo-contact shifts as constraints for MD refinement of solution structures of paramagnetic metalloproteins.

Additional Material: 4 Ill.