BIOC 4580 Lecture Notes - Lecture 11: Bacteriorhodopsin, X-Ray Crystallography, Protein Structure
22 Mar 2018
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Document Summary
Nomenclature of transmembrane proteins is based on their topology. May need to knockout endogenous cysteines, then insert cysteines by site-specific mutagenesis. Cysteine residues have free thiol groups and are highly reactive, can be inserted anywhere in a protein by site-directed mutagenesis, then labels at specific sites by maleimide derivative. A cys-less construct of the protein is made by replacing all the naturally existing cys residues by ala (conservative), constructs usually still functional. Cys residues are the introduced one at a time into specific loops/turns within the cys-less protein, using site-directed mutagenesis of the existing amino acid. Cys residues react covalently with various thiol-specific reagents, such as maleimide derivatives. Both membrane-permeant and membrane-impermeant maleimides are available, with tags to allow detection. Each protein construct is expressed in intact cells, reacted with a permeant and impermeant maleimide, and its location inferred. Cys may be unreactive if located in a tightly folded region of the protein and inaccessible to reagent.
