Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein.
Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.
Vinogradova
O
O
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Badola
P
P
Czerski
L
L
Sönnichsen
F D
FD
Sanders
C R
CR
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002845:Chromatography / Q000379:methods; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003902:Detergents; D019852:Diacylglycerol Kinase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008823:Micelles; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012996:Solutions; D012997:Solvents
D003432:Cross-Linking Reagents; D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles; D012996:Solutions; D012997:Solvents; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase