PMID:11697728
| Citation |
Alexandrovich, A, Czisch, M, Frenkiel, TA, Kelly, GP, Goosen, N, Moolenaar, GF, Chowdhry, BZ, Sanderson, MR and Lane, AN (2001) Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain. J. Biomol. Struct. Dyn. 19:219-36 |
|---|---|
| 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 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). 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 deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 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. |
| Links |
PubMed Online version:10.1080/07391102.2001.10506734 |
| Keywords |
Amino Acid Sequence; Calorimetry, Differential Scanning; Circular Dichroism; DNA Helicases/chemistry; DNA Helicases/genetics; DNA Repair; Dimerization; Endodeoxyribonucleases/chemistry; Endodeoxyribonucleases/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Protein Denaturation; Protein Structure, Quaternary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Solutions; Static Electricity; Thermodynamics |
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