Deletion of a single helix from the transmembrane domain causes large changes in membrane insertion properties and secondary structure of the bacterial conjugation protein TrwB
| Autor: | Igor de la Arada, Rosa L. Segura, Ana J. Vecino, Félix M. Goñi, Itziar Alkorta, José Luis R. Arrondo, Fernando de la Cruz |
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| Rok vydání: | 2012 |
| Předmět: |
Membrane lipids
Lipid Bilayers Mutant Biophysics Biology Biochemistry Protein Structure Secondary Transmembrane domain Cell membrane Membrane Lipids Escherichia coli medicine Membrane protein orientation Lipid bilayer Protein secondary structure Infrared spectroscopy Sequence Deletion Escherichia coli Proteins Vesicle Cell Membrane Cell Biology Membrane protein reconstitution Conjugative coupling protein DNA-Binding Proteins Tetrahydrofolate Dehydrogenase stomatognathic diseases medicine.anatomical_structure Membrane protein Liposomes Mutation human activities |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 0005-2736 |
| DOI: | 10.1016/j.bbamem.2012.08.015 |
| Popis: | 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, TrwBΔN50 that lacks one of the two α-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 TrwBΔN70 lacking the whole TMD. Interestingly, when TrwBΔN50 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 TrwBΔN70, 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. © 2012 Elsevier B.V. This work was supported with funds from the Spanish Ministerio de Educación y Ciencia (Grant No. BFU2007-62062) and the Basque Government (Grant No. IT-461-07). A.J.V. was a postdoctoral scientist supported by the University of the Basque Country. R.L.S. was a postdoctoral scientist supported by Basque Government. Work in the de la Cruz laboratory was supported by Spanish Ministry of Education (BFU2011-26608). |
| Databáze: | OpenAIRE |
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