Direct Observation of Downhill Folding of λ-Repressor in a Microfluidic Mixer
Open Access
- 1 September 2009
- journal article
- research article
- Published by Elsevier BV in Biophysical Journal
- Vol. 97 (6), 1772-1777
- https://doi.org/10.1016/j.bpj.2009.07.003
Abstract
The protein λ6-85 has been implicated in barrierless folding by observations of kinetic relaxation after nanosecond T-jump. In this work we observed folding of this protein after dilution of a high denaturant in an ultrarapid microfluidic mixer at temperatures far below the thermal midpoint. The observations of total intensity and spectral shift of tryptophan fluorescence yielded distinctly different kinetics and activation energies. These results may be explained as diffusion on a low-barrier, one-dimensional, free-energy surface, with different probes having different sensitivities along the reaction coordinate. Additionally, we observed an extremely fast phase within the mixing time that was not observed by T-jump, suggesting that the ensemble of unfolded states populated at high denaturant is distinct from those accessible at high temperature.Keywords
Funding Information
- Burroughs Wellcome Fund
- National Science Foundation (PHY 0120999, EF-0623664)
- U.S. Department of Energy (DE-AC52-07NA27344)
This publication has 30 references indexed in Scilit:
- Dynamics of one-state downhill protein foldingProceedings of the National Academy of Sciences of the United States of America, 2009
- An experimental survey of the transition between two-state and downhill protein folding scenariosProceedings of the National Academy of Sciences of the United States of America, 2008
- Tuning λ6–85 Towards Downhill Folding at its Melting TemperatureJournal of Molecular Biology, 2007
- Optimization of a Microfluidic Mixer for Studying Protein Folding KineticsAnalytical Chemistry, 2006
- Kinetics are probe-dependent during downhill folding of an engineered λ 6–85 proteinProceedings of the National Academy of Sciences of the United States of America, 2005
- Rate−Temperature Relationships in λ-Repressor Fragment λ6-85 FoldingBiochemistry, 2004
- Folding at the speed limitNature, 2003
- Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in MicrosecondsPhysical Review Letters, 1998
- Diffusive dynamics of the reaction coordinate for protein folding funnelsThe Journal of Chemical Physics, 1996
- Funnels, pathways, and the energy landscape of protein folding: A synthesisProteins-Structure Function and Bioinformatics, 1995