NMR Characterization of Membrane Protein−Detergent Micelle Solutions by Use of Microcoil Equipment

Abstract

Using microcoil NMR technology, the uniformly 2 H, 15 N-labeled integral membrane protein OmpX and the phosphocholine derivative detergent Fos-10 (n-decylphosphocholine), we investigated solutions of mixed protein-detergent micelles to determine the influence of the detergent concentration on the NMR spectra of the protein. In a first step, we identified key parameters that influence the composition of the micelle solutions, which resulted in a new protocol for the preparation of well-defined concentrated protein solutions. This led to the observation that highquality 2D [ 15 N, 1 H]-TROSY spectra of OmpX reconstituted in mixed micelles with Fos-10 were obtained only in a limited range of detergent concentrations. Outside of this range from about 90 mM to 180 mM, we observed a significant decrease of the average peak intensity. Relaxationoptimized NMR measurements of the rotational and translational diffusion coefficients of the OmpX/ Fos-10 mixed micelles, D r and D t , respectively, then showed that the stoichiometry and the effective hydrodynamic radius of the protein-containing micelles are not significantly affected by high Fos-10 concentrations, and that the deterioration of NMR spectra is due to the increased viscosity at high detergent concentrations. The paper thus provides a basis for refined guidelines on the preparation of integral membrane proteins for structural studies.

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

“…5A, bottom), indicating that the protein is motionally restricted with correlation times slower than the solution NMR time scale. This result is consistent with the vast majority of membrane protein NMR studies where detergent concentrations well above the CMC are needed to ensure the presence of a single protein molecule per micelle [52, 54, 55]. …”

“…2A, S5, [40]), as previously reported for two other outer membrane proteins, OmpX and OmpW [52, 53]. Ail in 170 mM DePC, at 45°C, yields a well-resolved 1 H/ 15 N correlation spectrum with narrow, homogenous resonance linewidths and large chemical shift dispersions, consistent with an overall ordered fold and relatively uniform backbone dynamics across the entire protein sequence.…”

“…High detergent concentrations well above the CMC are needed to ensure the presence of no more than one protein or protein complex per micelle, which is essential for maintaining sufficiently rapid correlation times for solution NMR [52, 54]. This effect may be understood in terms of the probability distribution of protein molecules in micelles, with the probability P(i) of finding i proteins per micelle given by: $$\mathrm{normalP}false(\mathrm{normali}false)={\mathrm{n}}^{\mathrm{i}}\left(\frac{{\mathrm{e}}^{-\mathrm{normaln}}}{\mathrm{i}!}\right)$$ where n, the total number of proteins per micelle, depends on the protein concentration (C protein ) and the micelle concentration (C micelle ) as: $$\mathrm{normaln}=\frac{{\mathrm{normalC}}_{\text{protein}}}{{\mathrm{normalC}}_{\text{micelle}}}$$ and C micelle depends on the detergent’s concentration (C detergent ), aggregation number (N a ) and CMC as: $${\mathrm{C}}_{\text{micelle}}=\frac{{\mathrm{C}}_{\text{detergent}}-\text{CMC}}{{\mathrm{normalN}}_{\mathrm{normala}}}$$…”

“…By contrast, above 80 mM DePC, P(i>1) decreases slowly down to near 0% at 200 mM. Above such high levels of detergents, the NMR spectral quality has been shown to decrease [52] as the deleterious effects of sample viscosity begin to counter the benefits of protein dispersion.…”

Characterization of Small Isotropic Bicelles with Various Compositions

“…(11) 60 ), which leads to line broadening and decrease in sensitivity in NMR spectra. According to an experimental NMR study 65 , the effect of the increased lipid concentration can be much 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 more severe than predicted by eq. (11), when the concentration of lipids exceeds 10% ( correspond to ca.…”