Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):18970-5. Epub 2007 Nov 19.

Combined kinetic and thermodynamic analysis of alpha-helical membrane protein unfolding.

Author information

  • 1Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. p.curnow@bristol.ac.uk

Abstract

The analytical toolkit developed for investigations into water-soluble protein folding has yet to be applied in earnest to membrane proteins. A major problem is the difficulty in collecting kinetic data, which are crucial to understanding any reaction. Here, we combine kinetic and thermodynamic studies of the reversible unfolding of an alpha-helical membrane protein to provide a definitive value for the reaction free energy and a means to probe the transition state. Our analyses show that the major unfolding step in the SDS-induced denaturation of bacteriorhodopsin involves a reduction in alpha-helical structure and proceeds with a large free-energy change; both our equilibrium and kinetic measurements predict that the free energy of unfolding in the absence of denaturant is +20 kcal.mol(-1), with an associated m-value of 25 kcal.mol(-1). The rate of unfolding in the absence of denaturant, k(u)(H(2)O), is surprisingly very slow ( approximately 10(-15) s(-1)). The kinetics also give information on the transition state for this major unfolding step, with a value for beta (m(f)/[m(f) + m(u)]) of approximately 0.1, indicating that the transition state is close to the unfolded state. We thus present a basis for mapping the structural and energetic properties of membrane protein folding by mutagenesis and classical kinetics.

PMID:
18025476
[PubMed - indexed for MEDLINE]
PMCID:
PMC2141892
Free PMC Article

Images from this publication.See all images (8)Free text

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Scheme 1
Scheme 2
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk