Protein chemistry, peptide mapping, and preliminary structural characterization of saccharomyces cerevisiae sterol c-24 methyltransferase expressed in escherichia coli
Soape, Matthew P
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At this time, no 3-dimensional structure of the Sterol C-24 Methyltransferase (SMT) enzyme has been discovered. Having such a representation of this enzyme, especially with a bound inactivator, will illustrate what contact amino acids and motifs are necessary for catalysis of a methyl transfer from S-adenosyl- L-methionine (AdoMet) to the C-24 position on the preferred substrate of zymosterol in the ergosterol biosynthetic pathway. In order to achieve this goal, protein chemistry techniques of Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE), Q-sepharose anion-exchange, and 26/60 SephacrylTM S-300 gel permeation chromatography were used to generate pure SMT for structure determination by our colleague Dr. David W. Christianson at the University of Pennsylvania. Quantification methods involving Bradford protein assay, Ultraviolet (UV) absorbance at 280 nm, activity assays with [methyl - 3H3] AdoMet, and Western blot analysis were developed to track the amount of total protein and active SMT from the yeast Wild Type (WT) and Y81W mutant throughout the purification process. Peptide mapping, using the mechanism-based inactivator [3-3H]26,27 dehydrozymosterol (DHZ), was also developed to independently locate motifs in the primary sequence associated with the active center. The main experimental findings are as follows: (1) From 5 g of Escherichia coli (E. coli) BL21 (DE3) host cell pellet harboring the pET23a(+) plasmid with the Y81W mutation was obtained 5 mg Fast Protein Liquid Chromatography (FPLC) pure recombinant Y81W mutant SMT-DHZ-AdoMet complex. (2) From a partial tryptic digest of a SMT-DHZ-AdoMet complex was obtained 9.1 mg of DHZ bound peptide. The identity of the peptide-DHZ complex was not resolved. (3) Using a sample of FPLC pure Y81W mutant SMT complexed with DHZ was attempted X-ray diffraction. Poor resolution crystals were obtained and did not diffract well. The results are interpreted to imply the approaches developed herein to purify SMT can be applied to further structural determination.