Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein
| Autor: | Begoña Ugarte-Uribe, Itsaso Hormaeche, Itziar Alkorta, Félix M. Goñi, Rosa L. Segura, Ana J. Vecino, Fernando de la Cruz, Sandra Águila |
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| Rok vydání: | 2010 |
| Předmět: |
Proteolipids
education Lipid Bilayers Protein reconstitution Biophysics Biology Biochemistry Substrate Specificity Adenosine Triphosphate R388 Adenine nucleotide Escherichia coli Nucleotide Lipid bilayer Adenosine Triphosphatases chemistry.chemical_classification Nucleotides Escherichia coli Proteins Nucleotide binding protein Vesicle Cell Biology Flow Cytometry Relaxosome Conjugative coupling protein DNA-Binding Proteins Bacterial conjugation Transmembrane domain Membrane protein chemistry Conjugation Genetic |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 0005-2736 |
| DOI: | 10.1016/j.bbamem.2010.07.005 |
| Popis: | Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV couplingprotein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The proteinspecificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity. This work was supported with funds from the Spanish Ministerio de Educación y Ciencia (grant no. BFU2007-62062), from the Diputación Foral de Bizkaia (DIPE07/16) and from LSHM-CT-2005_019023 (European VI Framework Program). R.L.S. was a postdoctoral scientist supported by a CSIC I3P postdoctoral fellowship. A.J.V. and S.A. were pre-doctoral students supported by the Basque Government; B.U.U. was pre-doctoral student supported by the University of the Basque Country. |
| Databáze: | OpenAIRE |
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