Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain

Alexander Alexandrovich, Michael Czisch, Thomas A. Frenkiel, Geoffrey P. Kelly, Nora Goosen, Geri F. Moolenaar, Babur Z. Chowdhry, Mark R. Sanderson, Andrew N. Lane

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E.coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5±1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 Å) at protein concentrations greater than 4 μM to about 18 Å at concentrations less than 0.1 μM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 °C at 36 μM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with δH1=23 kJ mol-1, K1(298)=0.4 μM and δH2=184 kJ mol-1. This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol-1 and a van't Hoff enthalpy change of 354 kJ mol-1, confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed.

Original languageEnglish
Pages (from-to)219-236
Number of pages18
JournalJournal of Biomolecular Structure and Dynamics
Volume19
Issue number2
DOIs
StatePublished - 2001

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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