Chemical structures of MNG amphiphiles (MNG-1, MNG-2, and MNG-3) and their linear counterparts (MPA-1, MPA-2, MPA-3, MPA-4, DM, UDM, DDM, and TDM). The critical micelle concentration (CMC) value for each agent, measured via hydrophobic dye solubilization, is indicated in parentheses.

SDS-12% PAGE analysis and Western blot detection of MelB. MelB samples were subjected to SDS-PAGE analysis, and MelB was detected by Western blotting using anti-His tag antibody. 10 μg membrane proteins were applied for the untreated membrane (memb.) or detergent extracts prior to ultracentrifugation (−), and equal volume of solutions were loaded for samples that the ultracentrifugation were applied (+).

Long-term stability of LeuT and R. capsulatus superassembly in MNG amphiphiles or conventional detergents. (a) Time course of activity ([³H]-Leu binding) assay for LeuT solubilized with MNG amphiphiles (MNG-1, MNG-2, and MNG-3) and DDM at 0.026 wt % above its critical micelle concentration (CMC) (total concentrations: 0.035 wt % DDM, 0.028 wt % MNG-1, 0.027 wt % MNG-2 and 0.027 wt % MNG-3). LeuT activity was monitored at indicated time points, using a scintillation proximity assay (SPA), for protein stored at the room temperature. Results are expressed as % activity relative to the appropriate day 0 measurement. Normalized results are expressed as mean ± s.e.m. (n = 2). (b) Time course of stability of R. capsulatus superassembly purified with MNG amphiphiles (MNG-1, MNG-2, and MNG-3) and conventional detergents (MPA-3 and DDM) at 1 × CMC. The absorption ratios (A₈₇₅/A₆₈₀) of the detergent or amphiphile samples were followed as a function of time.

GPCR stability in MNG amphiphiles or conventional detergents. (a) T_m values of the β₂AR-T4L plotted in terms of wt % of the MNG amphiphiles (MNG-1, MNG-2, and MNG-3) or conventional detergents (MPA-1, MPA-3, DM, DDM, and TDM). β2AR-T4L with bound carazolol (an inverse agonist) was incubated with various agents at the various concentrations at indicated temperatures for 5 min prior to fluorescence emission measurements. Normalized results are expressed as mean ± s.e.m. (n = 3, 4, or 5). (b) Specific activities (pmol/mg) of M₃AchR in DDM and MNG-3. The activity of the protein was evaluated after being washed and eluted with buffer including DDM or MNG-3, but without CHS, via a binding assay involving the antagonist [³H] N-methyl scopolamine, in the absence (t = 0 hr, − CHS; the first bar) or presence of CHS (t = 0 hr, + CHS; the second bar). The DDM- and MNG-3-purified M₃AchR samples were stored at 4 °C for 15 hours, and then activities were measured again in the presence of CHS (t = 15 hr, + CHS; the third bar). Results are expressed as mean ± s.d. (n = 3).

Stability of SQR solubilized with MNG amphiphiles or conventional detergents. (a) CPM assays for SQR solubilized with MNG amphiphiles (MNG-1, MNG-2, and MNG-3) or conventional detergents (MPA-4, DDM, DM and SDS) at 10 × CMC. The unfolding of the each protein was monitored at 40 °C for 130 min using a microplate spectrofluorometer. Gel filtration analysis of SQR in (b) DDM or (c) MNG-3 at 10 × CMC. SQR in DDM or MNG-3 was incubated for 120 min at 40 °C. (d) Time course of SQR activity in MNG-3 or DDM. Each agent was used at 10 × CMC (0.01 wt % for MNG-3, 0.087 wt % for DDM) and 50 × CMC (0.05 wt % for MNG-3, 0.44 wt % for DDM). Note that 50 × CMC MNG-3 is comparable to DDM at 10 × CMC in terms of wt %. The catalytic rate constant (k_cat) is plotted as a function of incubation time. Data at t = 0 correspond to the activity of SQR following thermal activation performed at 30 °C for 20 min. Protein solubilized with each agent was incubated at 40 °C for a further 120 min, and activity of the protein was measured at the designated times. The k_cat values at each time point were calculated by analyzing reaction data according to Michaelis-Menten kinetics.

Image and x-ray diffraction pattern from crystals of cytochrome b₆f /MNG-3 complexes. X-ray diffraction by cytochrome b₆f crystal obtained in the presense of MNG-3. The left panel represents a portion of the pattern (0.5 degree oscillation range). Resolution limits are marked with arrows (the white cross is due to the tiling of the detector). Top right: enlargement of the red square with two strong spots near the resolution limit. A section through the two strong spots is shown in the lower right corner.