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Lipid Bilayer Composition Modulates the Unfolding Free Energy of a Knotted Α-Helical Membrane Protein

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Lipid Bilayer Composition Modulates the Unfolding Free Energy of a Knotted Α-Helical Membrane Protein Lipid bilayer composition modulates the unfolding free PNAS PLUS energy of a knotted α-helical membrane protein M. R. Sandersa, H. E. Findlaya, and P. J. Bootha,1 aDepartment of Chemistry, King’s College London, SE1 1DB London, United Kingdom Edited by David Baker, University of Washington, Seattle, WA, and approved January 12, 2018 (received for review August 18, 2017) α-Helical membrane proteins have eluded investigation of their gregation, most helical proteins are thought to insert directly into thermodynamic stability in lipid bilayers. Reversible denaturation the membrane cotranslationally with the assistance of the curves have enabled some headway in determining unfolding free translocon apparatus. Although the exact mechanism of mem- energies. However, these parameters have been limited to deter- brane protein insertion is unclear, the final stages of protein gent micelles or lipid bicelles, which do not possess the same me- folding are most likely to occur in the lipid bilayer (4, 5). Equally, chanical properties as lipid bilayers that comprise the basis of lipids can induce posttranslational repositioning of helices and natural membranes. We establish reversible unfolding of the protein orientation within the membrane (6, 7). If the unique membrane transporter LeuT in lipid bilayers, enabling the compar- folded state is an equilibrium structure, then it should be possible ison of apparent unfolding free energies in different lipid composi- to achieve the folded structure via other pathways, as has been demonstrated for example from coexpression (8), from reas- tions. LeuT is a bacterial ortholog of neurotransmitter transporters sembly of protein fragments (9), or from cell-free synthesis (10). and contains a knot within its 12-transmembrane helical structure. Moreover, it has proved possible to regain the folded structure of Urea is used as a denaturant for LeuT in proteoliposomes, resulting some membrane proteins from a partly denatured state in urea, in the loss of up to 30% helical structure depending upon the lipid by refolding directly into liposomes (11). Additionally, the sta- bilayer composition. Urea unfolding of LeuT in liposomes is revers- bility of some membrane proteins is such that they can be suc- ible, with refolding in the bilayer recovering the original helical cessfully refolded in detergent micelles or mixed detergent/lipid structure and transport activity. A linear dependence of the unfold- mixtures (11–13). These latter systems have enabled insight into ing free energy on urea concentration enables the free energy to be the thermodynamic stability of membrane proteins by translating extrapolated to zero denaturant. Increasing lipid headgroup charge the classical chemical denaturation approach of water-soluble BIOPHYSICS AND or chain lateral pressure increases the thermodynamic stability of proteins studies. A folded protein is denatured and a reversible COMPUTATIONAL BIOLOGY LeuT. The mechanical and charge properties of the bilayer also af- refolding reaction established such that the free energy of fect the ability of urea to denature the protein. Thus, we not only unfolding can be determined between the folded and chemically gain insight to the long–sought-after thermodynamic stability of an unfolded state. The initial approach for helical membrane pro- α-helical protein in a lipid bilayer but also provide a basis for studies teins used a partially denatured state in SDS and established an of the folding of knotted proteins in a membrane environment. equilibrium with a folded state in renaturing detergent micelles (14). The best-studied case is bacteriorhodopsin (bR) for which protein folding | membrane proteins | protein–lipid interactions folding rates and yields have been shown to be dependent on detergent, lipid dynamics, detergent/lipid concentrations, and lipid bilayer mechanics (3, 15–22). As a result, the unfolded state nowledge of the energetics of protein reactions is necessary actually changes over time and thus is not at equilibrium. A Kto generate physical descriptions of cellular processes. The comprehensive equilibrium and kinetic study overcame this and thermodynamic stability of a protein is a fundamental parameter. established a pseudoequilibrium over a specific time period; A third of all cellular proteins reside in membranes, and those nonetheless, extensive extrapolation to zero denaturant was re- with helical structures are ubiquitous across prokaryotes and eu- quired to attain a free energy of unfolding in the absence of SDS karyotes. The hydrophobic character of these proteins together (23, 24). More recently, a novel steric trapping approach was with their native membrane surroundings can make them experi- mentally challenging to study (1). As a result, there is little in- Significance formation on key properties, including folding from their primary amino sequences to the final functional structure. Several lines of Cells in our bodies sense and communicate with the outside evidence suggest that these functional states of native integral world via proteins embedded in membranes that surround the membrane proteins are equilibrium structures (2). However, to cells. As with all proteins, a fundamental parameter governing date there are no thermodynamic measurements of the helical their biological function is the inherent, thermodynamic stability membrane protein class in lipid bilayers. The few thermodynamic of the folded state. Surprisingly, there is no measure of this stability measurements that exist for helical membrane proteins thermodynamic stability in a lipid membrane for the ubiquitous are in detergent micelles or bicelles, which do not adequately class of membrane proteins with structures based on α-helices. reproduce the properties of the lipid membrane that surrounds We remedy this through the study of a protein related to the the proteins in cells. Evidence has emerged that charge and me- physiologically important membrane proteins that are partly chanical properties of the bilayer play an important role in the responsible for transmitting signals in the nervous system. We folding, stability, and function of helical proteins (3). Such prop- identify key properties of the surrounding lipid membrane that erties cannot be reproduced in micelles and only to a very limited regulate the thermodynamic stability of the protein. extent in bicelles. Studies are therefore needed in lipid bilayers to determine the extent to which the fold of a membrane protein is Author contributions: M.R.S., H.E.F., and P.J.B. designed research; M.R.S. performed re- dictated by the amino acid sequence and the surrounding lipid search; M.R.S. analyzed data; and M.R.S. and P.J.B. wrote the paper. membrane. This not only advances membrane protein folding The authors declare no conflict of interest. studies, by providing boundaries in which natural folding operates, This article is a PNAS Direct Submission. but gives a physical basis for factors that alter membrane protein Published under the PNAS license. stability and misfolding linked to disease. 1To whom correspondence should be addressed. Email: [email protected]. The hydrophobicity of membrane proteins means they have a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. high propensity to aggregate in aqueous solutions. To avoid ag- 1073/pnas.1714668115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1714668115 PNAS Latest Articles | 1of10 Downloaded by guest on October 2, 2021 used, which employs different assumptions for equilibrium ther- modynamic determination and avoids denaturants, instead using streptavidin binding to the doubly biotinylated protein and in- ducing partial unfolding (25). Urea has also proven useful in de- naturing helical membrane proteins (11) and has moved the folding field forward to larger, more dynamic proteins; namely the 12-transmembrane (TM) major facilitator superfamily (MFS) (12, 26). These proteins are partially denatured by urea, and although the exact regions of the protein that are denatured are currently unknown, it seems likely that urea accesses aqueous exposed re- gions and those accessible via the substrate translocation site (11, 12, 26). Far-UV CD provides a categorical measure of the partly denatured state in terms of secondary structure loss. To demon- strate correct refolding, we combine regain of function, coupled with recovery of helical structure. Here, we extend chemical denaturation approaches on mem- brane transporter proteins to free-energy measurements in lipid bilayers. At present, there have been no successful measure- ments of the thermodynamic stability of helical membrane pro- teins in bilayers. There have been thermodynamic studies of TM Fig. 1. Crystal structure of LeuT in OG (PDB ID code 3GJD) taken from Quick helix oligomerization in bilayers, using a variety of methods et al. (39). A shows the full structure of the protein with the knot core based on steric trapping (27), cross-linking (28), FRET (29), and highlighted in blue, the slipknot loop in yellow, and the slipknot tail in green TOXCAT (based on Tox-R mediated activation of a chloram- from opposite viewpoints (B) taken from sequence data presented by knotprot database. phenicol acetyltransferase gene) (30, 31). The equilibrium chemical denaturation approach we use here, enables us to probe the influ- ence of the mechanical and charge properties of the lipid bilayer that key groundwork for further studies on how knots form at a mo- are vital to the folding and
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