Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein

Hydrogen-deuterium exchange can be 3 sensitive indicator of protein structu ral integrity. Comparisons were made between cerebroside sulfate activator protein (CSAct) in the native state and after treatment with guanidine hydr ochloride plus dithiothreitol, Native protein has three internal disulfide bonds and treated protein has no internal disulfide bonds. The comparisons were made using hydrogen-deuterium exchange measured by electrospray ioniza tion mass spectrometry, percentage alpha-helical content measured by circul ar dichroism and biological activity measured by the ability to support ary lsulfatase A-catalyzed sulfate hydrolysis from cerebroside sulfate. In acid ic solvent native protein has 59 exchange refractory protons and treated pr otein has 20 exchange refractory protons (44 and 14% of the exchangeable pr oton populations, respectively). In native protein the size of the exchange refractory proton population is sensitive to changes in pH, temperature an d the presence of a ligand. It is uninfluenced by the presence or absence o f glycosyl groups attached to Asn21. Helical content is virtually identical in native and treated protein. Biological activity is significantly reduce d but not obliterated in treated protein. The hydrogen-deuterium exchange p rofile appears to be a sensitive signature of the correctly folded protein, and reflects a dimension of the protein structure that is not apparent in circular dichroic spectra or in the ability of the protein to support aryls ulfatase A-catalyzed sulfate hyrolysis from sulfatide, The hydrogen-deuteri um exchange profile will be a valuable criterion for characterizing mutant forms of CSAct produced by recombinant and synthetic paradigms and also the native and mutant forms of related proteins, Copyright (C) 2000 John Wiley Sr Sons, Ltd.