Synthesis and conformational studies of a transmembrane domain from a diverged microsomal Delta(12)-desaturase.
Transmembrane domains of the acyl-coenzyme A and acyl phosphatidylcholine-utilizing desaturases may control interactions with electron transport domains, be involved in substrate specificity and/or serve as a structural foundation for the enzyme. To experimentally define these domains and as a prelude to detailed NMR studies, a segment of the microsomal Delta(12)-desaturase/acetylenase CREP-1 predicted to contain the amino-proximate transmembrane domain TM-A was chemically synthesized. A modified 9-fluorenylmethoxycarbonyl procedure was used that ensured complete deprotections at each homologation and the peptide was purified in good yield by reverse-phase high-performance liquid chromatography. Conformational studies of the hydrophobic peptide TM-A demonstrated its strong propensity for folding into an alpha-helical secondary structure. The helical content was 58-65% in aqueous solutions containing 40-80% 2,2,2-trifluoroethanol, a lipomimetic solvent, and was maximal at low temperatures. The peptide assumed a largely helical character when incorporated into phospholipid bilayers and detergent micelles. Experimental evidence is in agreement with neural network predictions that a transmembrane domain exists between residues R-44 and I-67 in this diverged Delta(12)-desaturase.
Minto
Robert E
RE
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA. mintore@muohio.edu
Gibbons
William J
WJ
Cardon
Thomas B
TB
Lorigan
Gary A
GA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D044943:Fatty Acid Desaturases / Q000737:chemistry* / Q000145:classification; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D008861:Microsomes / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water / Q000737:chemistry
D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; D014867:Water; D014270:Trifluoroethanol; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase