ACTIVE BARNASE VARIANTS WITH COMPLETELY RANDOM HYDROPHOBIC CORES

The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core. If some measure of exquisite, na tive-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes , either by design or by natural or experimental evolution. To test th e minimum requirements for a core to provide sufficient structural int egrity for enzymatic activity, we have produced mutants of the ribonuc lease barnase in which 12 of the 13 core residues have together been r andomly replaced by hydrophobic alternatives. Using a sensitive biolog ical screen, we find that a strikingly high proportion of these mutant s (23%) retain enzymatic activity in vivo. Further substitution at the 13th core position shows that a similar proportion of completely rand om hydrophobic cores supports enzyme function. Of the active mutants p roduced, several have no wild-type core residues. These results imply that hydrophobicity is nearly a sufficient criterion for the construct ion of a functional core and, in conjunction with previous studies, th at refinement of a crudely functional core entails more stringent sequ ence constraints than does the initial attainment of crude core functi on. Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contribute d by the hydrophobic core would greatly reduce the initial hurdle on t he evolutionary pathway to novel enzymes. Similarly, experimental deve lopment of novel functional proteins might be simplified by limiting c ore design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.