A study of the structure, folding and assembly of diacylglycerol kinase, an integral membrane protein

Prokaryotic diacylglycerol kinase is a 14 kDa, homotrimeric integral membrane protein located in the cytoplasmic membrane of gram-negative bacteria. It is a largely helical protein with three transmembrane spanning domains and two amphipathic helices at the membrane/water interface. There are three active sites per trimer situated at subunit interfaces. DAGK functions as part of the membrane derived oligosaccharide pathway which gram-negative bacteria employ when stressed by conditions of high osmolarity.; As an integral membrane protein, DAGK has not yet lent itself to structural characterization using classical techniques (i.e. NMR). Therefore, a non-classical approach, engineered thiol crosslinking was employed to map the oligomeric interface of the protein. Single-cysteine mutants spanning DAGK's transmembrane segments and part of a cytoplasmic domain were crosslinked under several different conditions. Based on the results from these studies, residues 53 and 75 appear to be in stable proximity within the DAGK homotrimer while position 52 is more distal. This establishes the critical structural information that TM 2 of DAGK forms the central three-fold symmetry axis within the trimeric protein. Furthermore, in lipid vesicles and mixed micelles DAGK executes motions that are not present in native membranes. Mobility is also higher for DAGK in mixed micelles compared to POPC vesicles.; The overall kinetics of membrane insertion and folding of DAGK were also examined. Lipid/detergent-free denaturant stock and micellar stock solutions were diluted into model membranes and the rates and efficiencies of folding/insertion were monitored. Productive insertion of DAGK from denaturant solutions into mixed micelles occurred more rapidly than into lipid vesicles, suggesting that the rate limiting step for DAGK assembly is bilayer traversal. Furthermore, efficiency of productive folding/insertion was found to be dependent upon the nature of the initial structural state of the protein with there being a strong inverse correlation between folding efficiency and the degree of protein denaturation within the stock enzyme solution. We also identified one single cysteine mutant, Y16C which appears to be a critical site in the folding pathway of DAGK. The methods developed in both the structural and folding studies should be generally applicable to other membrane proteins.