Solvent‐tuning the collapse and helix formation time scales of λ _*

The λ_6‐85^* pseudo‐wild type of lambda repressor fragment is a fast two‐state folder (k_f ≈ 35 μsec⁻¹ at 58°C). Previously, highly stable λ_6‐85^* mutants with k_f > 30 μsec⁻¹ have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune λ_6‐85^* away from two‐state folding. We test this prediction by examining the folding thermodynamics and kinetics of λ_6‐85^* in a stabilizing solvent, 45% by weight aqueous ethylene glycol at −28°C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X‐ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low‐barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two‐state folding of λ_6‐85^* is not a robust process.