A Survey of λ Repressor Fragments from Two-State to Downhill Folding

We survey the two-state to downhill folding transition by examining 20 λ_6-85^* mutants that cover a wide range of stabilities and folding rates. We investigated four new λ_6-85^* mutants designed to fold especially rapidly. Two were engineered using the core remodeling of Lim and Sauer, and two were engineered using Ferreiro et al.'s frustratometer. These proteins have probe-dependent melting temperatures as high as 80 °C and exhibit a fast molecular phase with the characteristic temperature dependence of the amplitude expected for downhill folding. The survey reveals a correlation between melting temperature and downhill folding previously observed for the β-sheet protein WW domain. A simple model explains this correlation and predicts the melting temperature at which downhill folding becomes possible. An X-ray crystal structure with a 1.64-Å resolution of a fast-folding mutant fragment shows regions of enhanced rigidity compared to the full wild-type protein.