By: Desiree Morris, Thomas Yi, Angela Schlegel

 A group from spring 2011 ran a SDS PAGE gel on stage four AP. They found lower bands that could be DsbC.  When mass spec was run on the stage four enzyme, they found a protein that could be DsbC.  This could explain why pure enzyme from Sigma Aldrich is more thermally stable than stage four AP.

 The Dsb family of proteins catalyze the formation of double bonds.  They are located in the periplasm of the E coli.  Kurokawa et al concluded that overexpression of DsbC stabilizes proteins with multiple double bonds.  DsbC is a dimeric protein with a monomeric MW of 23.3kDa  It fuctions as an isomerase and chaperone.

 Fructose-bisphosphate aldolase confers no likely stabilization  Cystine transporter subunit also likely not stabilizing

 Determine identities of ~24.7 kDa and ~35.1 kDa proteins in the stage 4 sample Hypothesis The lower molecular weight band is believed to be DsbC or one of the other Dsb proteins The higher MW protein is predicted to have a role in stabilizing/forming disulfide bonds or another thermal stability function

Concentrate Stage 4 Enzymes SDS-PAGE => Coomassie => De-stain In-gel trypsin digest Mass spectroscopy: LC/MS-MS (ESI)

R f = band distance/dye front distance

Sample 1: 24.7 kDa

Sample 2: 35.1 kDa

 Additional sample submission.  2 bands were included in each sample. A greater amount needs to be added for better analysis  Run another gel. Larger Pore size for expansion of protein cluster in the 30.6 kDa region. Further investigation of the proteins through MS/MS  Why? DsbC is believed to be reoxidized by an uncharacterized protein acting as a disulfide isomerase (STRING).  Protein-protein interactions  Determine locations of protein interactions which could lead to a proposed method.

 Kurokawa, Yoichi, Hideki Yanagi, and Takashi Yura. "Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia Coli." Applied and Environmental Microbiology(2000) 66.9:  Messens, Jori & Collet, Jean-François. “Pathways of Disulfide Formation in Escherichia coli” The International Journal of Biochemistry & Cell Biology(2006) 38:  "STRING: Functional Protein Association Networks." STRING: Functional Protein Association Networks. Web. 24 Apr