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 substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.
Yamasaki
R
R
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Hoshino
M
M
Wazawa
T
T
Ishii
Y
Y
Yanagida
T
T
Kawata
Y
Y
Higurashi
T
T
Sakai
K
K
Nagai
J
J
Goto
Y
Y
D000255:Adenosine Triphosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D002417:Cattle; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007782:Lactoglobulins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D012235:Rhodamines / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D055672:Static Electricity; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
D002232:Carbocyanines; D018834:Chaperonin 60; D004220:Disulfides; D007782:Lactoglobulins; D012235:Rhodamines; D012492:Salts; C085321:cyanine dye 5; C005358:tetramethylrhodamine; D000255:Adenosine Triphosphate; D013884:Thiosulfate Sulfurtransferase