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 ca n undergo autocatalytic activation and is subject to proteolytic self- inactivation. Mutant enzyme forms were prepared to eliminate the initi al autoprocessing site and thus form an active single-chain protein fo r structure-function studies. Two mutants of CMV proteinase were clone d and expressed in Escherichia coli. The A143V mutant was a conservati ve substitution at the first internal cleavage site. The S132A mutant modified one of the triad of residues responsible for catalytic activi ty. Through the use of computer-controlled high-cell-density fermentat ions the mutant proteins were expressed in E. coli at similar to 170 m g/L as both soluble (similar to 40% of total) and inclusion-body forms (similar to 60% of total). The soluble enzyme was purified by standar d methods; inclusion-body protein was isolated by standard methods aft er refolding and solubilization in guanidine or urea. Sedimentation eq uilibrium and sedimentation velocity analyses reveal that the enzyme u ndergoes concentration-dependent aggregation. It exhibits a monomer do uble left right arrow dimer equilibrium (K-d = 1 mu M) at low concentr ations and remains dimeric at high concentrations (28 mg/ml). Differen tial scanning calorimetry data for protein thermal unfolding fit best to a non-two-state model with two components (T-m = 52.3 and 55.3 degr ees C) which subsequently aggregate upon unfolding. Analysis of the sh ort-UV circular dichroism spectra of protein forms resulting from expr ession as soluble molecules (not refolded) reveals that the two mutant s have very similar secondary structures which comprise a mixed struct ural motif of 20% alpha-helix, 26% beta-sheet, and 53% random coil. Th ough soluble and active (A143V mutant only), CD analysis revealed that protein refolded from inclusion bodies did not exhibit spectra identi cal to that of protein expressed only in soluble form.