Functional in vitro assembly of the integral membrane bacterial thermosensor DesK.
The Bacillus subtilis DesK histidine kinase (HK) is an integral membrane thermosensor that forms part of a regulatory circuit which controls the physical state of membrane lipids. In the pursuit of biochemical and structural approaches to study lipid fluidity-dependent DesK thermosensing, we found that standard expression methods failed to produce enough amounts of a fully functional protein. Here, we describe a high-yield purification method based in an Escherichia coliin vitro transcription-translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 degrees C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.
Martín
Mariana
M
Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina.
Albanesi
Daniela
D
Alzari
Pedro M
PM
de Mendoza
Diego
D
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D000071677:Histidine Kinase; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D013696:Temperature
D001426:Bacterial Proteins; D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase