Halorhodopsin from natronomonas pharaonis forms a trimer even in the presence of a detergent, dodecyl-beta-D-maltoside.
Halorhodopsin (HR) is a transmembrane seven-helix retinal protein, and acts as an inward light-driven Cl(-) pump. HR from Natronomonas pharaonis (NpHR) can be expressed in Escherichia coli inner membrane in large quantities. Here, we showed that NpHR forms the trimer structure even in the presence of 0.1% (2 mm) to 1% (20 mm) dodecyl-beta-d-maltoside (DDM), whose concentrations are much higher than the critical micelle concentration (0.17 mm). This conclusion was drawn from the following observations. (1) NpHR in the DDM solution showed an exciton-coupling circular dichroism (CD) spectrum. (2) From the elution volume of gel filtration, the molecular mass of the NpHR-DDM complex was estimated. After evaluation of the mass of the bound DDM molecules, the mass of NpHR calculated was approximately equal to that of the trimer. (3) The cross-linked NpHR by glutaraldehyde gave the SDS-PAGE corresponding to the trimer. Mass spectra of these samples also support the notion of the trimer. Using the membrane fractions expressing NpHR (Escherichia coli and Halobacterium salinarum), CD spectra showed exciton-coupling, which suggests strongly the trimer structure in the cell membrane.
Sasaki
Takanori
T
Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
Kubo
Megumi
M
Kikukawa
Takashi
T
Kamiya
Masakatsu
M
Aizawa
Tomoyasu
T
Kawano
Keiichi
K
Kamo
Naoki
N
Demura
Makoto
M
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D005960:Glucosides / Q000737:chemistry*; D006216:Halobacteriaceae / Q000737:chemistry*; D025602:Halorhodopsins / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D013053:Spectrophotometry; D013696:Temperature
D003432:Cross-Linking Reagents; D005960:Glucosides; D025602:Halorhodopsins; C040358:dodecyl maltoside