Kinetic study of folding and misfolding of diacylglycerol kinase in model membranes.
Despite the relevance of membrane protein misfolding to a number of common diseases, our understanding of the folding and misfolding of membrane proteins lags well behind soluble proteins. Here, the overall kinetics of membrane insertion and folding of the homotrimeric integral membrane protein diacylglycerol kinase (DAGK) is addressed. DAGK was purified into lipid/detergent-free urea and guanidinium solutions and subjected to general structural characterization. In urea, the enzyme was observed to be monomeric but maintained considerable tertiary structure. In guanidinium, it was also monomeric but exhibited much less tertiary structure. Aliquots of these DAGK stock solutions were diluted 200-fold into lipid vesicles or into detergent/lipid mixed micelles, and the rates and efficiencies of folding/insertion were monitored. Reactions were also carried out in which micellar DAGK solutions were diluted into vesicular solutions. Productive insertion of DAGK from denaturant solutions into mixed micelles occurred much more rapidly than into lipid vesicles, suggesting that bilayer transversal represents the rate-limiting step for DAGK assembly in vesicles. The efficiency of productive folding/insertion into vesicles was highest in reactions initiated with micellar DAGK stock solutions (where DAGK maintains a nativelike fold and oligomeric state) and lowest in reactions starting with guanidinium stocks (where DAGK is an unfolded monomer). Moreover, the final ratio of irreversibly misfolded DAGK to reversibly misfolded enzyme was highest following reactions initiated with guanidinium stock solutions and lowest when micellar stocks were used. Finally, it was also observed that very low concentrations of detergents were able to both enhance the bilayer insertion rate and suppress misfolding.
Nagy
J K
JK
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Lonzer
W L
WL
Sanders
C R
CR
D000595:Amino Acid Sequence; D003902:Detergents / Q000737:chemistry; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005960:Glucosides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D015203:Reproducibility of Results; D012996:Solutions; D014461:Ultracentrifugation; D014508:Urea / Q000737:chemistry
D003902:Detergents; D005960:Glucosides; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D012996:Solutions; C040358:dodecyl maltoside; D014508:Urea; D019852:Diacylglycerol Kinase; D019791:Guanidine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine