ACCESS OF LIGANDS TO CAVITIES WITHIN THE CORE OF A PROTEIN IS RAPID

We have investigated the magnitude and timescale of fluctuations withi n the core of a protein using the exchange kinetics of indole and benz ene binding to engineered hydrophobic cavities in T4 lysozyme. The cry stal structures of variant-benzene complexes suggest that relatively l arge scale fluctuations (1-2 Angstrom) of backbone atoms are required for entry of these ligands into the cove. Nonetheless, these ligands e nter the cavities rapidly, with bimolecular rate constants of similar to 10(6)-10(7) M(-1) s(-1) and a low activation barrier, 2-5 kcal mol( -1) These results suggest that protein cores undergo substantial fluct uations on the millisecond to microsecond timescale and that entry of small molecules into protein interiors is not strongly limited by ster ic occlusion.