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Solvent-tuning the collapse and helix formation time scales of _6-85^*

¹ Department of Physics, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA
² Department of Physics, Kansai Medical University, 18-89 Uyama-Higashi, Hirakata 573-1136, Japan
³ BioCAT at Advanced Photon Source, BCPS Department, Illinois Institute of Technology, Illinois 60439, USA
⁴ Department of Chemistry, and Center for Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA

(RECEIVED March 30, 2006; FINAL REVISION June 20, 2006; ACCEPTED August 3, 2006)

The _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 _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 _6-85^* away from two-state folding. We test this prediction by examining the folding thermodynamics and kinetics of _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 _6-85^* is not a robust process.

Keywords: collapse; heterogeneous kinetics; cryosolvent; stopped flow

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