Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 43, Pages 18451-18456Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1005729107
Keywords
protein folding; thermodynamic stability; linear free-energy relationship
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Funding
- EU [201924 EDICT]
- Royal Society
- European Membrane Protein consortium, EMeP [LSHG-CT-2004-504601]
- Wellcome Trust [062164/Z/00/Z]
- BBSRC [BB/F013183/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/F013183/1] Funding Source: researchfish
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Understanding how an amino acid sequence folds into a functional, three-dimensional structure has proved to be a formidable challenge in biological research, especially for transmembrane proteins with multiple alpha helical domains. Mechanistic folding studies on helical membrane proteins have been limited to unusually stable, single domain proteins such as bacteriorhodopsin. Here, we extend such work to flexible, multidomain proteins and one of the most widespread membrane transporter families, the major facilitator superfamily, thus showing that more complex membrane proteins can be successfully refolded to recover native substrate binding. We determine the unfolding free energy of the two-domain, Escherichia coli galactose transporter, GalP; a bacterial homologue of human glucose transporters. GalP is reversibly unfolded by urea. Urea causes loss of substrate binding and a significant reduction in alpha helical content. Full recovery of helical structure and substrate binding occurs in dodecylmaltoside micelles, and the unfolding free energy can be determined. A linear dependence of this free energy on urea concentration allows the free energy of unfolding in the absence of urea to be determined as +2.5 kcal.mol(-1). Urea has often been found to be a poor denaturant for transmembrane helical structures. We attribute the denaturation of GalP helices by urea to the dynamic nature of the transporter structure allowing denaturant access via the substrate binding pocket, as well as to helical structure that extends beyond the membrane. This study gives insight into the final, critical folding step involving recovery of ligand binding for a multidomain membrane transporter.
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