A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

Abstract

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

Fig. 1. Chemical structures of newly prepared TRIS-derived triglucosides (TDTs) and neopentyl glycol-derived triglucosides (NDTs).

Fig. 2. Thermal denaturation profile of UapA protein purified in DDM and then exchanged into novel TDTs (a) and NDTs (b) at detergent concentrations of CMC + 0.04 wt%. Thermal stability of the protein was monitored by CPM assay performed at 40 °C for 120 min. The relative amounts of folded protein were normalized relative to the most destabilizing condition in this experiment, that is, protein denaturation in DDM after 2 h incubation. Mean standard deviations (n = 2) for DDM, TDT-C9, TDT-C10, TDT-C11, TDT-C12, NDT-C9, NDT-C10, NDT-C11 and NDT-C12 are 4.9, 9.3, 2.3, 5.8, 6.1, 2.7, 9.4, 10.2, 9.5, respectively.

Fig. 3. Thermosolubility and functional profiles of detergent-solubilised MelB_St. The solubility test at elevated temperatures was carried out as described in the ESI. (a) Solubilised materials after ultracentrifugation of detergent-treated membranes were analysed by SDS-15%PAGE and Western blot. The total amount of MelB_St protein used in each assay is shown by the untreated membrane sample (Memb). (b) Histogram of band density. The solubilisation efficiency of MelB_St is expressed as a percentage of band density relative to the untreated membrane sample. The density was measured by ImageQuant software. Error bars, SEM, n = 2–4. (c) Galactoside binding. Right-side-out (RSO) membrane vesicles containing MelB_St or MelB_Ec were solubilised with DDM or NDT-C11 as described in the ESI. After ultracentrifugation, the supernatant was used to test melibiose reversal of Trp to dansyl-2-galacotside (D²G) FRET. Note the difference in FRET response of the D²G bound MelB to melibiose or water addition at the 2 min point.

Fig. 4. Long-term activity of wild type leucine transporter (LeuT), ligand binding affinity and KI accessibility of the LeuT E192C^TMR. Long-term stability was measured by using the transporter solubilized in novel amphiphiles (TDT-C11, TDT-C12, NDT-C11 and NDT-C12) and a conventional detergent (DDM). The detergents were used at CMC + 0.04 wt% (a) and CMC + 0.2 wt% (b). Protein activity for LeuT was measured by scintillation proximity assay (SPA). Results are expressed as % activity relative to activity at day 0 (mean ± s.e.m., n = 2). (c) Saturation binding of [³H]leucine assessed by SPA for mutant protein, LeuT E192C^TMR, in either CMC + 0.04 wt% DDM or NDT-C11. Data are fitted to a single site model. Data points are means ± s.e.m. with n = 3–4. (d) K_SV values were plotted as a function of leucine concentration at CMC + 0.04 wt% detergent concentration. A conventional detergent (DDM), newly prepared NDT-11, and previously reported MNG-3 were used for comparison. Data points are means ± s.e.m. with n = 3–4.