Dual-targeted labeling of proteins using cysteine and selenomethionine residues.
A new strategy for dual site-selective labeling of proteins that uses metabolically incorporated selenomethionine as a target for covalent modification by iodoacetamide derivatives, forming selenonium salts, is described. In the absence of free cysteine, labeling is specific and efficient. Dual-targeted labeling of a protein can be achieved with combinations of unique cysteine and methionine residues, if the cysteine is labeled first with a maleimide or another reagent that does not react with the selenomethionine. The method should be useful in biophysical applications such as fluorescence energy transfer.
Lang
Shenhui
S
Department of Chemistry, University of Waterloo, Waterloo, Ont., Canada N2L 3G1.
Spratt
Donald E
DE
Guillemette
J Guy
JG
Palmer
Michael
M
D001426:Bacterial Proteins / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics; D003545:Cysteine / Q000737:chemistry*; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006460:Hemolysin Proteins; D007460:Iodoacetamide / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012645:Selenomethionine / Q000737:chemistry*
C032162:AEDANS-calmodulin; D001426:Bacterial Proteins; C039092:CAMP protein, Streptococcus; D002147:Calmodulin; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D008301:Maleimides; D009282:Naphthalenesulfonates; D011506:Proteins; C043592:maleimide; D012645:Selenomethionine; D003545:Cysteine; D007460:Iodoacetamide