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When reduction leads to expansion part 2: on‐going undergraduate laboratory investigations on the roles of disulfide bonds within E. coli alkaline phosphatase
Author(s) -
Hazzard James T.,
Koussa Mounir A.,
Cornelison Matthew J.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.577.3
Subject(s) - dithiothreitol , tcep , chemistry , proteolysis , enzyme , alkaline phosphatase , disulfide bond , phosphatase , biochemistry , phosphine , catalysis
As a continuation of a guided inquiry undergraduate biochemistry laboratory experience we present data obtained on the investigation of the specific roles played by two disulfide bonds within E. coli alkaline phosphatase during thermal denaturation. Using a combination of CD and fluorescence (tryptophan and ANS) spectroscopy, enzyme activity assays, and limited tryptic proteolysis we show the dramatic effect of reduction of disulfide bonds on structural intergrity, enzymatic activity, and the susceptibility of the enzyme towards tryptic digestion. Interestingly, not only does each disulfide bond play a specific role in maintaining structural integrity, but the individual bonds can be selectively reduced with a specific reducing agent. Therefore, we present evidence that the individual effects of triscarboxyethyl phosphine (TCEP) and dithiothreitol (DTT) are quite different on the above mentioned enzymatic properties. Being a segment of a typical undergraduate biochemistry laboratory course, these investigations represent special projects for individual laboratory groups who make oral presentations to the entire class, providing a holistic approach to investigating the relationship between protein structure and function. This work supported in part by a grant from NSF (DUE 0837398 to JTH).