Effect of Detergent Concentration on Multidimensional Solution NMR Spectra of Membrane Proteins in Micelles

Two‐Dimensional 1H‐NMR of Transmembrane Peptides from Escherichia Coli Phosphatidylglycerophosphate Synthase in Micelles

“…This preparation resulted in an isotropic micellar solution that incorporated approximately 1 peptide/micelle ; the aggregation number of SDS micelles in pure water is about 75 (Cabane et al, 1985). The detergent concentration has been shown to affect NMR spectra of proteins and peptides in micelles (McDonnell and Opella, 1993). Adequate detergent concentrations (20-100-fold the critical micelle concentration) are required to fully solubilize the peptides and form uniform micelles.…”

Structure Determination of Membrane Proteins by NMR Spectroscopy

“…Protein-containing micelles reorient rapidly enough to give isotropic spectra, as shown in Figure 1A. However, the reorientation occurs slowly compared to that for soluble proteins of similar molecular mass, and as a result, micelle samples are very demanding and require careful sample preparation, 45 the use of high-field NMR spectrometers, and elevated temperatures. At that, many of the most informative solution NMR experiments cannot be applied to these samples because the short relaxation times result in the loss of signals during extended pulse sequences.…”

“…There is a long history to preparing samples of membrane-associated peptides and proteins in micelles for solution NMR studies. 45,[73][74][75][76] For all polypeptides, regardless of length or hydrophobicity, we use four different lipids (SDS (sodium dodecyl sulfate), DPC (dodecyl phosphatidyl choline), DHPC (dihexanoyl phophatidyl choline), LMPC (lyso myristoyl phosphatidyl choline)) in an initial screening of conditions for two-dimensional HSQC spectra of uniformly 15 N labeled samples. With the increasing level of activity in the field of solution NMR of membrane proteins, many new suggestions for lipids and combinations of lipids are emerging.…”

“…Any doubling or anomalous broadening of resonances is a warning sign that the polypeptide is aggregated or improperly folded. 45 We do not proceed with structural studies by solution NMR or even with sample preparation in bilayers or bicelles for solid-state NMR until these problems are resolved. This is a crucial checkpoint.…”

Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles

“…Suitable micelles for such studies must ensure the structural and functional integrity of the membrane protein, should be a good mimic of the natural environment in the cell membrane, and need to be sufficiently small to allow rapid Brownian motions of the mixed micelles in solution (e.g., refs. [10][11][12]. The large size of the structures obtained upon reconstitution and solubilization of membrane proteins in detergent micelles actually has limited the application of solution NMR techniques to such systems because of the slow tumbling in solution and the concomitantly large linewidths.…”