Probing enzyme quaternary structure by combinatorial mutagenesis and selection

Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to invest igate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme. By inserting random loops of four to seven residu es into a dimeric chorismate mutase and selecting for functional variants b y genetic complementation, we have obtained and characterized both monomeri c and hexameric enzymes that retain considerable catalytic activity. The lo w percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our exper iments suggest further that protein stability can be an important driving f orce in the evolution of oligomeric proteins.