Protein chemistry, peptide mapping, and preliminary structural characterization of saccharomyces cerevisiae sterol c-24 methyltransferase expressed in escherichia coli

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Title: Protein chemistry, peptide mapping, and preliminary structural characterization of saccharomyces cerevisiae sterol c-24 methyltransferase expressed in escherichia coli
Author: Soape, Matthew P
Abstract: 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 .
URI: http : / /hdl .handle .net /2346 /17951
Date: 2007-08

Citation

Protein chemistry, peptide mapping, and preliminary structural characterization of saccharomyces cerevisiae sterol c-24 methyltransferase expressed in escherichia coli. Master's thesis, Texas Tech University. Available electronically from http : / /hdl .handle .net /2346 /17951 .

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