CONFORMATIONAL PROPERTIES OF RHODOBACTER-CAPSULATUS CYTOCHROME C(2) WILD-TYPE AND SITE-DIRECTED MUTANTS USING HYDROGEN-DEUTERIUM EXCHANGE MONITORED BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

The conformational properties of Rhodobacter capsulatus cytochrome c(2 ) wild-type and two site-directed mutants (glycine 34 replaced by seri ne and proline 35 replaced by alanine) were characterized by their cha rge state distributions and hydrogen/deuterium (H/D) exchange properti es monitored by electrospray ionization mass spectrometry. The results suggest the presence of structural perturbations in the mutated cytoc hromes, an observation that is in agreement with their decreased confo rmational stabilities. In addition, a fast enzymatic procedure was dev eloped to identify regions for which the II-bonding or solvent accessi bility properties were perturbed by the mutations. In this procedure, deuterated peptides were separated and analysed by using liquid chroma tography directly coupled to the electrospray ionization source in ord er to minimize the occurrence of back-exchange during analysis. In the case of G34S, mutational effects were found for peptides 1-26, 38-51, 52-59 and 109-116, which in the Rb. capsulatus cytochrome c(2) struct ure correspond to extensive regions on the same side of the molecule a s the proximal histidine, as well as part of the C-terminal helix. In the case of P35A, mutational effects were found for peptides 1-26, 27- 37, 38-51 and 52-59, which in the Rb. capsulatus cytochrome c(2) stuct ure correspond to extensive regions on the same side of the molecule a s the proximal histidine. We show that the present set of mass spectro metric experiments is useful as an initial characterization of mutant conformational properties because the analyses require only nanomole q uantities of protein and can be performed rapidly.