A novel method of production and biophysical characterization of the catalytic domain of yeast oligosaccharyl transferase.
Oligosaccharyl transferase (OT) is a multisubunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of the Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, and a robust but novel method of production of the pure C-terminal domain of Stt3p at 60-70 mg/L in Escherichia coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well-dispersed two-dimensional (1)H-(15)N HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The replacement of a key residue, Asp(518), located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures, as demonstrated by CD, fluorescence and NMR spectroscopies. This observation strongly suggests that Asp(518) plays a critical structural role, in addition to the previously proposed catalytic role. Moreover, the activity of the protein was confirmed by saturation transfer difference and nuclear magnetic resonance titration studies.
Huang
Chengdong
C
Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
Mohanty
Smita
S
Banerjee
Monimoy
M
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006602:Hexosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008565:Membrane Proteins; D008823:Micelles; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D012967:Sodium Dodecyl Sulfate; D018698:Glutamic Acid; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae