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Continuing Studies on the Structural Stability of E. coli Alkaline Phosphatase by an Undergraduate Biochemistry Laboratory Course
Author(s) -
Hazzard James T.,
Hailey Alexander,
Klein Mary E.,
Korte Stepahnie R.,
Mason Ashley M.,
Narendran Nirushan H.,
Schnoebelen Carly L.
Publication year - 2012
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.26.1_supplement.619.3
Subject(s) - chemistry , denaturation (fissile materials) , biochemistry , alkaline phosphatase , proteolytic enzymes , enzyme , nuclear chemistry
On‐going investigations into the roles of the two disulfide bonds in E. coli alkaline phosphatase (AP) on structural stability and limited proteolytic degradation have been incorporated into a guided inquiry portion of an undergraduate biochemistry laboratory course. We studied the effects of reducing agents (DTT and TCEP) on the thermal and chemical (urea and guanidinium hydrochloride) denaturation of AP using a combination of tryptophan fluorescence and CD spectroscopy, also investigating the roles of Hofmeister series ions during thermal and chemical denaturation. We also present evidence for potential chaperonin‐like proteins found in the periplasmic lysates that contribute to the dramatic thermal stability of AP, relative to the highly purified commercial enzyme and used tandem mass spectrometry to identify likely stabilizing proteins. Being a portion of an undergraduate biochemistry laboratory course, these investigations represent data obtained from individual laboratory groups, that make oral presentations at the end of their research, providing a more real world approach to the educational experience. This work is supported, in part, by a grant from NSF (DUE 0837398).