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

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, N-15 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 Angstrom) at protein concentrations greater than 4 muM to about 18 Angstrom at concentrations less than 0.1 muM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (T. ca. 71.5 degreesC at 36 muM). 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 DeltaH(1 approximate to)23 kJ mol(-1), KI(298)=0.4 muM and DeltaH(2)=184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the then-nal 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. N-15 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.