Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein.
While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason.
Gorzelle
B M
BM
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA.
Nagy
J K
JK
Oxenoid
K
K
Lonzer
W L
WL
Cafiso
D S
DS
Sanders
C R
CR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000737:chemistry; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D017510:Protein Folding; D014508:Urea
D003432:Cross-Linking Reagents; D003902:Detergents; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; D010767:Phosphorylcholine; C028810:dodecylphosphocholine; D014508:Urea; D019852:Diacylglycerol Kinase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine