ON THE RELATIONSHIP BETWEEN THE METABOLIC AND THERMODYNAMIC STABILITIES OF T4 LYSOZYMES - MEASUREMENTS IN ESCHERICHIA-COLI

Escherichia coli cells were transformed with plasmids encoding 12 site -specific variants of T4 lysozyme, and pulse-chase protocols were used to measure the metabolic stability of each protein. The resulting hal f-lives ranged from 1 h to more than 50 h. Although the metabolic half -lives of T4 lysozymes correlated roughly with their thermal stabiliti es, three mutant enzymes were clear exceptions. A reasonably temperatu re-resistant variant, G156D, exhibited a half-life of 1 h. By contrast , two temperature-sensitive variants, T157I and I3G, were as metabolic ally stable as wild-type T4 lysozyme. Degradation of two short lived v ariants, L91P and G156P, required ATP both in vivo and in vitro. Degra dation of variant and wild-type enzymes was unimpaired in cells lackin g the Lon protease, Clp A or Clp P. However, degradation of L91P and G 156D was inhibited in Clp B-cells. Decreased proteolysis of L91P was a ccompanied by its accumulation in inclusion bodies, indicating that Cl p B prevents accumulation of aggregated protein either by preventing a ggregation of misfolded polypeptides or solubilizing aggregates.