Unfolding free energy of a two-domain transmembrane sugar transport protein

Abstract

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 þ2.5 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.protein folding | thermodynamic stability | linear free-energy relationship

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?…”

Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of <italic>Clematis montana</italic> lectin

“…Effect of denaturants on hemagglutinating activity and fluorescence spectrum of CML Urea, SDS, and GuHCl are widely used as common denaturants to study the structure -function relationships of protein [24]. As shown in Fig.…”

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity

“…Since the level of expression of the amplified protein appeared higher in membranes coming from cells grown in minimal medium than in the rich media, minimal medium was used for all further cell cultures. The appearance at *36 kDa in SDS-PAGE gels instead of the expected 54 kDa is a phenomenon routinely observed with membrane proteins (Ward et al, 2000;Ma et al, 2008Ma et al, , 2013Rath et al, 2009;Findlay et al, 2010;Bettaney et al, 2013), probably a result of only partial unfolding of a membrane protein in SDS (Findlay et al, 2010).…”