Single molecular observation of the interaction of GroEL with substrate proteins

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled sub strate proteins at the single molecule level by total internal reflection f luorescence microscopy. GroEL with a A133C mutation in the equatorial domai n was labeled with a fluorescent dye, tetramethylrhodamine. As substrate pr oteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The com plexes formed by GroEL with these substrates were characterized by size-exc lusion gel chromatography. The recovered complexes were then observed by fl uorescence microscopy. For both substrates, agreement of the fluorescent sp ots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by si ze-exclusion chromatography and microscopic binding by the fluorescence mic roscopy was done for the folding intermediate of Cy5-labeled bovine rhodane se. The fluorescence microscopy opens a new avenue for studying the interac tion of GroEL with substrate proteins. (C) 1999 Academic Press.