Initial characterization of autoprocessing and active-center mutants of CMV proteinase

Human cytomegalovirus (CMV) encodes a unique serine proteinase that is required in the maturation of the viral capsid. The CMV proteinase can undergo autocatalytic activation and is subject to proteolytic self-inactivation. Mutant enzyme forms were prepared to eliminate the initial autoprocessing site and thus form an active single-chain protein for structure-function studies. Two mutants of CMV proteinase were cloned and expressed inEscherichia coli. The A143V mutant was a conservative substitution at the first internal cleavage site. The S132A mutant modified one of the triad of residues responsible for catalytic activity. Through the use of computer-controlled high-cell-density fermentations the mutant proteins were expressed inE. coli at 170mg/L as both soluble (40% of total) and inclusion-body forms (60% of total). The soluble enzyme was purified by standard methods; inclusion-body protein was isolated by standard methods after refolding and solubilization in guanidine or urea. Sedimentation equilibrium and sedimentation velocity analyses reveal that the enzyme undergoes concentration-dependent aggregation. It exhibits a monomerdimer equilibrium (K _d =1M) at low concentrations and remains dimeric at high concentrations (28 mg/ml). Differential scanning calorimetry data for protein thermal unfolding fit best to a non-two-state model with two components (T _m =52.3 and 55.3c) which subsequently aggregate upon unfolding. Analysis of the short-UV circular dichroism spectra of protein forms resulting from expression as soluble molecules (not refolded) reveals that the two mutants have very similar secondary structures which comprise a mixed structural motif of 20%-helix, 26%-sheet, and 53% random coil. Though soluble and active (A143V mutant only), CD analysis revealed that protein refolded from inclusion bodies did not exhibit spectra identical to that of protein expressed only in soluble form.