Site-directed mutagenesis of GroEL: Developing a system for monitoring allosteric movements by fluorescence resonance energy transfer
by Yang, Yu, M.S., UNIVERSITY OF MARYLAND, COLLEGE PARK, 2006, 82 pages; 1439194

Abstract:

The Escherichia coli chaperonin protein GroEL can assist protein folding to its native state through the consumption of ATP. Accompanying this process, GroEL undergoes structural change, resulting in an expansion of the central cavity. Monitoring apical domain movement by fluorescence resonance energy transfer (FRET) between two mobile apical fluorophores, can provide information about the GroEL allosteric transitions. To reach this goal, the three native cysteine residues on each subunit of wild type GroEL were removed and a new cysteine site in the apical domain was introduced by site-directed mutagenesis. Fluorescent probes were attached to the cysteine residues, allowing us to perform FRET experiments. The observed change of FRET efficiency (E) reported the GroEL structural changes.

 
AdviserGeorge H. Lorimer
SchoolUNIVERSITY OF MARYLAND, COLLEGE PARK
SourceMAI/ 45-02, p. , Feb 2007
Source TypeThesis
SubjectsBiochemistry
Publication Number1439194
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