Barriers to protein folding: Formation of buried polar interactions is a slow step in acquisition of structure

In the Mn mutant of the Are repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The Mn refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength, Formation of the, wild-type salt bridges ina hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations, The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer, The second step is postulated to be rate limiting for wild type, Replacing the salt bridge with hydrophobic interactions lowers this barrier for Mn, This makes the first kinetic barrier rate limiting for Mn refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.