BARNASE, BINASE, AND THEIR HYBRIDS - DIFFERENCES IN CONFORMATION AND HEAT DENATURATION PARAMETERS

Differential scanning microcalorimetry and circular dichroism were use d to elucidate the conformational and heat-denaturation features of ba rnase (Ba), binase (Bi), and their hybrids (1-25)Ba/(26-110)Bi, (1-72) Ba/(73-110)Bi, (1-25)Ba/(26-72)Bi/(73-110)Ba, and (1-72)Bi/(73-110)Ba constructed by an original method of ''homolog recombination.'' Heat d enaturation of barnase, binase, and their hybrids at pH 5.5 and 2.4 is a two-state transition. The denaturation temperature of binase is hig her than that of barnase; the lower the pH, the larger the difference. The hybrids and barnase also differ in thermostability. Reduction in a-helicity with pH lowered from 5.5 to 2.4 is accompanied by a marked drop in the melting temperature (22-25 degrees C). The differences in a-helicity of the ribonucleases are related to alterations in the seco nd and third a-helices. The mutation-induced changes in the free energ y of denaturation of barnase deduced from thermal and urea denaturatio n nearly coincide. Simultaneous substitutions have an additive effect on barnase thermostability. The barnase and binase regions determining the thermostability of a hybrid relative to the original proteins are specified.