Relative Domain Folding and Stability of a Membrane Transport Protein

Abstract

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of ~ +2 kcal.mol-1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C terminal domain exhibits wild type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of +2 kcal.mol-1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains. 1 Relative domain folding and stability of a membrane transport proteinHarris et al. Response to referees' commentsWe thank the referees for their helpful comments and have made changes to the manuscript in accordance with the issues raised by the editor and referees as detailed below. Additionally to improve clarity we have made colour version of the figures. EditorWe have re-written the abstract and expanded the explanation of the single Trp mutants, p2&5.It is hard to comment on the absolute magnitude of the free energy value determined for LacY and GalP, in the absence of thermodynamic measurements on other membrane proteins as well as studies using different methods to assess thermodynamic stability in lipid bilayers and kinetic stability. The free energy is small implying that the inherent protein structure has relatively low thermodynamic stability. It could be that the bilayer increases the kinetic stability, or that the absolute magnitude is partly dependent on the renaturing solvent since it is hard to separate the folding systems from the solvent surroundings in membrane protein unfolding studies. Referee 1Technical concerns: 1. DDM CMC in ureaThe CMC of DDM in different concentrations of urea was measured and showed that micelles are present with a DDM concentration of 1mM even at the highest urea concentration of 8M (as previously used for GalP). We have clarified this in the methods (p15) and added a figure S8 to the supplementary information showing the increase in DDM CM...

In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers

“…42. Briefly, LacY with a C-terminal 10-His tag was overexpressed from the pET28a vector in BL21-AI E. coli (Life Technologies).…”

Circular dichroism spectroscopy of membrane proteins

“…37 However, denaturants are not universally effective for all integral membrane proteins, and thus conditions must be determined experimentally for each protein.…”

The safety dance: biophysics of membrane protein folding and misfolding in a cellular context

“…Interestingly, conformational stability measurements of diacylglycerol kinase (DAGK) (Lau & Bowie, 1997), bacteriorhodopsin (bR) (Curnow & Booth, 2007), the KcsA potassium channel (Barrera et al, 2008), and aquaglyceroporin GlpF (Veerappan et al, 2011) have suggested large thermodynamic separation of the native and denatured reference states (Δ G unf = 16 to 31 kcal/mol). On the other hand, studies of disulfide bond reducing protein B (DsbB) (Otzen, 2003), galactose transporter GalP (Findlay et al, 2010), lactose permease LacY (Harris et al, 2014), human peripheral myelin protein 22 (PMP22) (Schlebach et al, 2013), and the rhomboid protease (Baker & Urban, 2012) have suggested a more modest thermodynamic preference for their native conformations (Δ G unf = 0 to 4.5 kcal/mol). Can it be that the range of thermodynamic stabilities of single domain wild-type α-helical membrane proteins varies by more than an order of magnitude?…”