Mass spectrometric measurement of changes in protein hydrogen exchange rates that result from point mutations.

Point mutations, as well as additions or deletions of entire domains, are frequently produced to study protein function; however, to infer function from mutant proteins, it is imperative that their structural integrity be verified. Although detailed structural studies can be performed by using NMR or crystallography, for practical reasons mutant proteins usually are characterized by using less rigorous techniques. Here it is shown that measurement of hydrogen exchange rates via electrospray ionization mass spectrometry is a sensitive and generally applicable method for detection of conformational or dynamic changes that result from point mutations. Hydrogen exchange experiments were performed on a bacterial phosphocarrier protein (HPr) and two variants produced by conversion of either serine-46 to aspartic acid (S46D) or serine-31 to alanine (S31A), where the differences in the ΔG of folding relative to the wild type were 1.5 and 0.5 kcal/mol, respectively. Whereas no significant differences were found for the intact mutant and wild-type proteins, changes in deuterium incorporation could be detected within specific regions produced by peptic proteolysis of the deuterium-labeled proteins. Thus, energetically small changes in conformation (or dynamics) that result from point mutations can be characterized by mass spectrometric measurements of hydrogen exchange rates. Furthermore, these changes can be localized to specific regions within the protein.