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, 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.
Alexandrovich
A
A
Randall Centre for Molecular Mechanisms of Cell Function, New Hunt's House, King's College, London, UK.
Czisch
M
M
Frenkiel
T A
TA
Kelly
G P
GP
Goosen
N
N
Moolenaar
G F
GF
Chowdhry
B Z
BZ
Sanderson
M R
MR
Lane
A N
AN
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D055672:Static Electricity; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D012996:Solutions; C058093:UvrB protein, E coli; D004706:Endodeoxyribonucleases; C066567:UvrC protein, E coli; D004265:DNA Helicases