A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation.
In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.
Department of Biochemistry and Molecular Biology, Darwin Building, University College London, Gower Street, London WC1E 6BT, UK.
Yie Chia
YC
Lee
Rashpal
R
Flora
James A
JA
McCafferty
Jayesh
J
Gor
Irina R
IR
Tsaneva
Stephen J
SJ
Perkins
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D009166:Mycobacterium leprae / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D012965:Sodium Chloride / Q000737:chemistry; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D002021:Buffers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012965:Sodium Chloride; C071059:Holliday junction DNA helicase, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases