Gerard H. M. Huysmans a,b, Stephen A. Baldwin a,b, David J. Brockwell a,c,1, and Sheena E. Radford a,c,1
-Author Affiliations
aAstbury Centre for Structural Molecular Biology,
bInstitute of Membrane and Systems Biology, and
cInstitute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom
Edited by Alan R Fersht, Medical Research Council Centre, University of Cambridge, Cambridge, United Kingdom, and approved November 18, 2009 (received for review October 15, 2009).
Abstract
Inspired by the seminal work of Anfinsen, investigations of the folding of small water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilize their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Here we use mutational analysis to describe the transition state involved in the reversible folding of the β-barrel membrane protein PhoPQ-activated gene P (PagP) from a highly disordered state in 10 M urea to a native protein embedded in a lipid bilayer. Analysis of the equilibrium stability and unfolding kinetics of 19 variants that span all eight β-strands of this 163-residue protein revealed that the transition-state structure is a highly polarized, partly formed β-barrel. The results provide unique and detailed insights into the transition-state structure for β-barrel membrane protein folding into a lipid bilayer and are consistent with a model for outer membrane protein folding via a tilted insertion mechanism.
beta barrel membrane protein PagP phi-value analysis protein folding
Footnotes
1To whom correspondence may be addressed. E-mail:d.j.brockwell@leeds.ac.uk or s.e.radford@leeds.ac.uk.
Author contributions: G.H.M.H., S.A.B., D.J.B., and S.E.R. designed research; G.H.M.H. performed research and analyzed data; and G.H.M.H., S.A.B., D.J.B., and S.E.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0911904107/DCSupplemental.
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