Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability

The mutation Glu108 --> Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of fu nction associated with the destabilizing substitution Leu99 --> Gly (L99G). Surprisingly, the two sites are 11 Angstrom apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand thi s result we have carried out a detailed thermodynamic, enzymatic and struct ural analysis of these mutant lysozymes as well as a related variant with t he substitution Leu99-->Ala. It was found that E108V does increase the stab ility of L99G, but it also increases the stability of both the wild-type pr otein and L99A by essentially equal amounts. The effects of E108V on enzyma tic activity are more complicated. The mutation slightly reduces the maxima l rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same tim e, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even th ough the double mutant L99G/E108V has a slightly lower maximal rate than L9 9G, over a period of 20-30 minutes it hydrolyzes more substrate. This decre ase in the rate of thermal inactivation appears to be the basis of the acti on of E108V as a second-site revertant of L99G. Mutant L99A creates a cavit y of volume 149 Angstrom(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 Angstrom, displacement of part of helix F (residues 108-11 3), creating a solvent-accessible declivity. In the double mutant, L99G/E10 8V, this helix returns to a position akin to wild-type, resulting in a cavi ty of volume 203 Angstrom(3). Whether the mutation Glu108 --> Val is incorp orated into either wild-type lysozyme, or L99A or L99G, it results in a dec rease in crystallographic thermal factors, especially in the helices that i nclude residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restrict ion of conformational fluctuation, provides a structural basis for the incr ease in thermostability. (C) 1999 Academic Press.