Circularization changes the folding transition state of the src SH3 domain

Native state topology has been implicated as a major determinant of protein -folding mechanisms. Here, we test experimentally the robustness of the src SH3-domain folding transition state to changes in topology by covalently c onstraining regions of the protein with disulfide crosslinks and then perfo rming kinetic analysis on point mutations in the context of these modified proteins. Circularization (crosslinking the N and C termini) of the src SH3 domain makes the protein topologically symmetric and causes delocalization of structure in the transition state ensemble suggesting a change in the f olding mechanism. In contrast, crosslinking a single structural element (th e distal beta -hairpin) which is an essential part of the transition state, results in a protein that folds 30 times faster, but does not change the d istribution of structure in the transition state. As the transition states of distantly related SH3 domains were previously found to be very similar, we conclude that the free energy landscape of this protein family contains deep features which are relatively insensitive to sequence variations but c an be altered by changes in topology. (C) 2001 Academic Press.