Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein

Abstract

Hydrogen–deuterium exchange can be a sensitive indicator of protein structural integrity. Comparisons were made between cerebroside sulfate activator protein (CSAct) in the native state and after treatment with guanidine hydrochloride 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 ionization mass spectrometry, percentage α‐helical content measured by circular dichroism and biological activity measured by the ability to support arylsulfatase A‐catalyzed sulfate hydrolysis from cerebroside sulfate. In acidic solvent native protein has 59 exchange refractory protons and treated protein has 20 exchange refractory protons (44 and 14% of the exchangeable proton populations, respectively). In native protein the size of the exchange refractory proton population is sensitive to changes in pH, temperature and the presence of a ligand. It is uninfluenced by the presence or absence of glycosyl groups attached to Asn21. Helical content is virtually identical in native and treated protein. Biological activity is significantly reduced but not obliterated in treated protein. The hydrogen–deuterium exchange profile 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 arylsulfatase A‐catalyzed sulfate hydrolysis from sulfatide. The hydrogen–deuterium 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 © 2000 John Wiley & Sons, Ltd.

Intrinsic Structural and Functional Determinants within the Amino Acid Sequence of Mature Pulmonary Surfactant Protein SP-B

“…Extensive reduction of disulfides in small proteins with compact folding requires substantial denaturation of the structure (34). This is the case, for instance, for proteins that are substantially homologous with SP-B such as the saposins or sulfate activator protein (35,36). No reductive treatment as severe as the one used in this work, including extensive denaturation of the protein checked by circular dichroism, has been previously carried out with SP-B.…”

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics

“…In previous studies, in vitro DTT reduction led only to minor qualitative losses in helicity at both acidic and neutral pH in the StPSI [4,33]. Reduced saposin B was observed to have a higher helix content after reduction (63.9% compared to 51.2% in the native protein) [21], however, the effects of purely disulfide bond reduction cannot be delineated from structural perturbation from the modification procedure. In this work, the secondary structure is instead reported as variation, or change in secondary structure, and was observed to fluctuate slightly throughout the protein sequences.…”

“…Over the past decades, disulfide reduction has been explored peripherally alongside other experiments on saposin and saposin-like proteins [4,5,7,[19][20][21][22]. When examined alongside these results, new insights are shed on their possible role in this diverse family of membraneactive proteins.…”

“…When re-calculated as a sum over the entire dimer, net variation remains relatively constant across the simulations (S1 Table). Saposin B has been the subject of hydrogen-deuterium exchange experiments performed with and without the three native disulfide bonds intact [21]. In this study, experiments on native saposin B yielded a total exchange of 77 to 80 protons, whereas disulfide reduced saposin B exchanged~125 (out of a total of 134 and 140 total theoretically exchangeable protons, respectively).…”