Porcine cerebroside sulfate activator (saposin B) secondary structure: CD, FTIR, and NMR studies.
Cerebroside sulfate activator protein (CSAct or saposin B) is one of a group of heat stable, low-molecular-weight proteins that appear to share a common structural motif. These have been referred to as saposin-like proteins and are thought to share a multiple amphipathic helical barrel structure with a conserved pattern of disulfide linkages. Porcine kidney CSAct was prepared in high purity and consisted of three major glycosylated subforms. The protein was studied by physical-chemical methods and evaluated by various methods for structural prediction. All suggest that CSAct has high amounts of alpha-helical conformation and little if any beta-sheet. Circular dichroism (CD) studies indicate 45-50% helical conformation depending on buffer and temperature. There was only a moderate loss in helical content with increasing temperature and no indication of thermal denaturation. Fourier transform infrared spectroscopy (FTIR) measurements on deuterium hydrated self-films also indicated a predominantly helical structure. Helical axis orientation was investigated by both oriented CD and FTIR dichroism, which suggested that the helical axes were roughly parallel and oriented along the axis of the surface on which the self-films had been deposited. One-dimensional nuclear magnetic resonance spectra showed large chemical shift dispersion, indicating a defined tertiary structure with little variation between 6 and 85 degrees C. NOESY spectra failed to show the strong NOE cross peaks expected for a highly helical conformation. This may indicate short-term conformational flexibility within the helices or molecular aggregation at the high protein concentrations employed. These observations are consistent with the 3-4-helix bundle motif suggested for saposin-like proteins by various predictive algorithms.
Waring
A J
AJ
Department of Pediatrics, Drew University-King Medical Center/University of California Los Angeles 90059, USA.
Chen
Y
Y
Faull
K F
KF
Stevens
R
R
Sherman
M A
MA
Fluharty
A L
AL
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D007668:Kidney / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D049231:Saposins; D017550:Spectroscopy, Fourier Transform Infrared; D049128:Sphingolipid Activator Proteins; D013552:Swine
D006023:Glycoproteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins