Three topologically equivalent core residues affect the transition state ensemble in a protein folding reaction

The single domain protein, interleukin-1β, is representative of a distinct class of proteins characterized by their β-trefoil topology. Each subdomain of this structural class is composed of a beta beta beta loop beta (βββLβ) motif comprised of ∼50 residues and gives the protein a pseudo- 3-fold axis of symmetry. A common feature of proteins in this topological family appears to be that they are slow folders, which reach the native state on the order of tens to 100s of seconds. Sequence analysis of interleukin-1β indicates that three phenylalanine residues located at positions 42, 101, and 146 are well conserved, separated by ∼50 residues in the primary sequence, located in similar positions in the pseudo-symmetric units of the trefoil, and are juxtaposed to one another in conformational space. These residues surround the hydrophobic cavity and “pin” the hairpin triplet cap to the core β-barrel. To determine if cap-barrel interactions are involved in maintaining the structural stability and cooperativity or in controlling the slow formation of the native state, we performed a series of mutational studies. The results indicate that interleukin-1β tolerates large increases in side-chain volume at these three topologically conserved sites with little effect on stability, while the kinetics show significant differences in both the unfolding and refolding rates. Taken together, our results indicate that these conserved core residues are essential contacts in the transition-state ensemble for folding.