Characterization of folding the four-helix bundle protein Rop by real-time NMR
1Department of Physical Chemistry and Institute of Biotechnology, University of Granada, 18071 Granada, Spain 2Oxford Center for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QT, UK and Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK 3Molecular Biophysics and Biochemistry 4Chemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA
5 To whom correspondence should be addressed. E-mail: lynne.regan{at}yale.edu
Rop is a four-helix bundle protein composed of two identical helix-loop-helix monomers. Protein folding monitored by stopped-flow fluorescence or CD exhibits biphasic kinetics when folding to low final denaturant concentrations. As the final concentration of denaturant is increased, the amplitude of the fast phase decreases, until at the highest concentrations the kinetics appear monophasic. We propose that the fast phase represents the formation of an intermediate. Here, we use real-time NMR to detect the formation of this intermediate and to characterize its structural features.
Keywords: protein folding/real-time NMR
Received November 27, 2007; accepted November 28, 2007.