Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins
1Departments of Chemistry, Yale University, Bass 322, 266 Whitney Avenue, New Haven, CT 06520-8114, USA 2Molecular Biophysics and Biochemistry, Yale University, Bass 322, 266 Whitney Avenue, New Haven, CT 06520-8114, USA 3Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, UK 4Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
3 To whom correspondence should be addressed. E-mail: lynne.regan{at}yale.edu
The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k0f = 29 s–1) and unfolds (k0u = 6 x 10–7 s–1) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.
Keywords: four-helix bundle/homologues/protein folding/protein stability/RNA-binding
Received December 6, 2007; revised December 6, 2007; accepted December 11, 2007.
5 Present address: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
6 Present address: Stanford University, School of Medicine, Stanford CA94306