Abstract
Hydrogen/deuterium exchange, which depends on solvent accessibility, can be probed by mass spectrometry (MS) to get information on protein conformation or protein–ligand interaction. In this work, the conformational properties of the cyanobacterium Anabaena wild-type ferredoxin as well as of two single-site mutants (Phe 65 Ala and Arg 42 Ala) were studied. After incubation of the wild type and mutant proteins in deuterated water and quenching of the exchange at low pH, the proteins were rapidly digested at high enzyme-to-substrate ratio using immobilized pepsin, and the resulting peptides were characterized using ESI-MS. We have identified specific regions for which the H-bonding or solvent accessibility properties were perturbed by the mutations. These results show that this approach can provide local information on the influence of mutations, even for a highly structured protein like ferredoxin, and sometimes in regions distant from the mutation point.
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Rémigy, H., Jaquinod, M., Pétillot, Y. et al. Probing the Influence of Mutations on the Stability of a Ferredoxin by Mass Spectrometry. J Protein Chem 16, 527–532 (1997). https://doi.org/10.1023/A:1026325914372
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DOI: https://doi.org/10.1023/A:1026325914372