Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volume 1818, Issue 12, Pages 3158-3166Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2012.08.015
Keywords
Membrane protein; Transmembrane domain; Membrane protein reconstitution; Membrane protein orientation; Infrared spectroscopy; Conjugative coupling protein
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Funding
- Spanish Ministerio de Educacion y Ciencia [BFU2007-62062]
- Basque Government [IT-461-07]
- University of the Basque Country
- Spanish Ministry of Education [BFU2011-26608]
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TrwB is an essential protein in the conjugative transfer of plasmid R388. The protein consists of a bulky cytosolic domain containing the catalytic site, and a small transmembrane domain (TMD). Our previous studies support the idea that the TMD plays an essential role in the activity, structure and stability of the protein. We have prepared a mutant, TrwB Delta N50 that lacks one of the two alpha-helices in the TMD. The mutant has been studied both in detergent suspension and reconstituted in lipid vesicles. Deletion of a single helix from the TMD is enough to increase markedly the affinity of TrwB for ATP. The deletion changes the secondary structure of the cytosolic domain, whose infrared spectroscopy (IR) spectra become similar to those of the mutant TrwB Delta N70 lacking the whole TMD. Interestingly, when TrwB Delta N50 is reconstituted into lipid membranes, the cytosolic domain orients itself towards the vesicle interior, opposite to what happens for wild-type TrwB. In addition, we analyze the secondary structure of the TMD and TMD-lacking mutant TrwB Delta N70, and found that the sum IR spectrum of the two protein fragments is different from that of the native protein, indicating the irreversibility of changes caused in TrwB by deletion of the TMD. (C) 2012 Elsevier B.V. All rights reserved.
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