Folding and self-assembly of herpes simplex virus type 1 thymidine kinase

Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postul ated to be a homodimer throughout the X-ray crystallography literature. Our study shows that HSV1 TK exists as a monomer-dimer equilibrium mixture in dilute aqueous solutions. In the presence of 150 mM NaCl, the equilibrium i s characterized by a dissociation constant of 2.4 muM; this constant was de termined by analytical ultracentrifugation and gel filtration experiments. Dimerization seems to be unfavorable for enzymantic activity: dimers show i nferior catalytic efficiency compared to e monomers. Moreover, soluble olig omers formed by self-assembly of the TK m the absence of physiological salt concentrations are even enzymatically inactive. This study investigates enzymatic and structural relevance of the TK dimer in vitro. Dissociation of the dimers into monomers is not accompanied by la rge overall changes in secondary or tertiary structure as shown by thermal and urea-induced unfolding studies monitored by circular dichroism and fluo rescence spectroscopy. A disulfide-bridge mutant TK (V119C) was designed be aring two cysteine residues at the dinner interface in order to crosslink t he two subunits covalently. Under reducing conditions, the properties of V1 19C and wild-type HSV1 TK (wt HSV1 TK) were identical in terms of expressio n yield, denaturing SDS PAGE gel electrophoresis, enzyme kinetics, CD spect ra and thermal stability. Crosslinked V119C (V119Cox) was found to have an increased thermal stability with a tm value of 59.1( +/-0.5)degreesC which is 16 deg. C higher than for the wild type protein. This is thought to be a consequence of the conformational restriction of the dimer interface. Furt hermore, enzyme kinetic studies on V119Cox revealed a K-m for thymidine of 0.2 muM corresponding to wt HSV1 TK, but a significantly higher Ka, for ATP . The present findings raise the question whether the monomer, not the dime r, might be the active species in vivo. (C) 2001 Academic Press.