Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein.
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 alpha-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.
Faull
K F
KF
Department of Psychiatry and Biobehavioral Sciences and the Neuropsychiatric Institute, UCLA, Los Angeles, California 90095, USA. faull@chem.ucla.edu
Higginson
J
J
Waring
A J
AJ
To
T
T
Whitelegge
J P
JP
Stevens
R L
RL
Fluharty
C B
CB
Fluharty
A L
AL
D000818:Animals; D002942:Circular Dichroism; D003903:Deuterium; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen; D006868:Hydrolysis; D066298:In Vitro Techniques; D007668:Kidney / Q000737:chemistry; D013058:Mass Spectrometry; D011487:Protein Conformation; D011489:Protein Denaturation; D011522:Protons; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013433:Sulfoglycosphingolipids / Q000378:metabolism; D013552:Swine
D006023:Glycoproteins; D011522:Protons; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013433:Sulfoglycosphingolipids; D006859:Hydrogen; D003903:Deuterium