Characterization of human cytomegalovirus protease dimerization by analytical centrifugation.
Human cytomegalovirus, a member of the herpesvirus family, encodes a maturational protease required for processing of the assembly protein and virus replication. The protease is synthesized as a precursor protein that undergoes autoproteolytic cleavage to yield a mature, 28-kDa enzyme. It has recently been demonstrated that mature human cytomegalovirus protease is capable of dimerization and that the dimer is the active species [Darke, P. L., Cole, J. L., Waxman, L., Hall, D. L., Sardana, M. K., & Kuo, L. C. (1996) J. Biol. Chem. 271, 7445-7449; Margosiak, S. A., Vanderpool, D. L., Sisson, W., Pinko, C., & Kan, C.-C. (1996) Biochemistry 35, 5300-5307]. Here, analytical equilibrium and velocity sedimentation measurements were used to define the thermodynamics of protease dimerization. Protease dimerization is well described by a homogeneous, reversible mass-action equilibrium. The apparent molecular weight of the protease decreases at higher protein concentrations, and good global fits to sedimentation equilibrium data require a positive value of the second virial coefficient, indicating that the protein exhibits thermodynamic nonideality. The magnitude of the nonideality is higher than expected on the basis of excluded volume and electrostatic effects and is not very sensitive to salt concentration, as would be expected for electrostatic effects. The dimer dissociation constants are in agreement with the values we previously determined by activity measurements and hydrodynamic techniques. Dimerization is enhanced by addition of glycerol or NaCl. The temperature dependence of the dimerization constant indicates that both delta H degree and delta S degree are negative, which is commonly observed in protein self-association reactions.
Cole
J L
JL
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
D002498:Centrifugation; D003587:Cytomegalovirus / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D012697:Serine Endopeptidases
D010450:Endopeptidases; D012697:Serine Endopeptidases; C071910:assemblin