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Electrostatic Potential Energy within a Protein Monitored by Metal Charge-Dependent Hydrogen Exchange

Janet S. Anderson ^*, David M. LeMaster  and Griselda Hernández 

^* Department of Chemistry, Union College, Schenectady, New York 12308-3107; and Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509

Correspondence: Address reprint requests and inquiries to G. Hernández, Tel.:518-474-4673; Fax: 518-473-2900; E-mail: griselda{at}wadsworth.org.

Hydrogen exchange measurements on Zn(II)-, Ga(III)-, and Ge(IV)-substituted Pyrococcus furiosus rubredoxin demonstrate that the log ratio of the base-catalyzed rate constants ( log k_ex) varies inversely with the distance out to at least 12 Å from the metal. This pattern is consistent with the variation of the amide nitrogen pK values with the metal charge-dependent changes in the electrostatic potential. Fifteen monitored amides lie within this range, providing an opportunity to assess the strength of electrostatic interactions simultaneously at numerous positions within the structure. Poisson-Boltzmann calculations predict an optimal effective internal dielectric constant of 6. The largest deviations between the experimentally estimated and the predicted pK values appear to result from the conformationally mobile charged side chains of Lys-7 and Glu-48 and from differential shielding of the peptide units arising from their orientation relative to the metal site.