Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein

Biochimica Et Biophysica Acta
Ana J VecinoItziar Alkorta

Abstract

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (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 protein specificity 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 ...Continue Reading

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Citations

Apr 2, 2013·Research in Microbiology·Minny BhattyPeter J Christie
Apr 9, 2013·Journal of Bacteriology·Eric CascalesPeter J Christie
Jul 23, 2013·Biophysical Journal·Dov LichtenbergFélix M Goñi
Aug 27, 2014·FEMS Microbiology Reviews·Elena CabezónIgnacio Arechaga
Sep 11, 2013·Biochimica Et Biophysica Acta·Rosa L SeguraItziar Alkorta
Jun 6, 2013·Biochimica Et Biophysica Acta·Rosa L SeguraItziar Alkorta
Dec 23, 2011·Current Opinion in Biotechnology·Elena CabezonIgnacio Arechaga
Jan 8, 2011·Biochimica Et Biophysica Acta·Ana J VecinoItziar Alkorta
Jun 10, 2017·Molecular Genetics and Genomics : MGG·Delfina LarreaMatxalen Llosa
Aug 28, 2020·Frontiers in Molecular Biosciences·Itxaso Álvarez-RodríguezItziar Alkorta

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