Micelle-assisted protein folding. Denatured rhodanese binding to cardiolipin-containing lauryl maltoside micelles results in slower refolding kinetics but greater enzyme reactivation.
Unfolded (inactive) rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) can be reactivated in the presence of detergents, e.g. lauryl maltoside (LM). Here, we report the reactivation of urea-unfolded rhodanese in the presence of mixed micelles containing LM and the anionic mitochondrial phospholipid, cardiolipin (CL). Reactivation times increased as the number of CL molecules/micelle was increased. A maximum of 94% of the activity was recovered at 2.2 CL/micelle. Only 71% of the activity was recovered in the absence of CL. The major zwitterionic mitochondrial phospholipid, phosphatidylcholine (PC), had no effect on the LM-assisted reactivation of rhodanese. Size exclusion chromatography showed that denatured, but not native, rhodanese apparently binds to micellar amounts of LM and CL/LM, but not to PC/LM micelles. The lifetime of the enzyme-micelle complex increased with the number of CL molecules/micelle. Furthermore, chromatographic fractions containing micelle-bound enzyme had no activity, while renatured rhodanese-containing fractions were active. These results suggest that transient complexes form between enzyme and both LM and CL/LM micelles, and that this complex formation may be necessary for reactivation. For CL/LM micelles, interactions may occur between the positively charged amino-terminal sequence of rhodanese and the negatively charged CL phosphate. Finally, this work shows that there are similarities between "micelle-assisted" and chaperonin-assisted rhodanese refolding.
Zardeneta
G
G
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Horowitz
P M
PM
D000818:Animals; D002308:Cardiolipins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005960:Glucosides; D007700:Kinetics; D008823:Micelles; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D002308:Cardiolipins; D005960:Glucosides; D008823:Micelles; C040358:dodecyl maltoside; D013884:Thiosulfate Sulfurtransferase