Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis

Abstract

A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.

Magnetically Oriented Bicelles with Monoalkylphosphocholines: Versatile Membrane Mimetics for Nuclear Magnetic Resonance Applications

“…The addition of lanthanides to bicelles has been shown to flip the orientation of the bilayer normal from perpendicular to parallel to the magnetic field 35 . Using this strategy, alignment flipping has been demonstrated with the D14PC/12PC system by 31 P solidstate NMR 31 . We have verified that it was also possible for D16PC/14PC (Figure 3) and D16PC/12PC (data not shown); the possibility to flip the bicelles thus appears to be a general property of MAPCHO bicelles.…”

“…Samples were then submitted to about 10 cycles of freeze (liquid N 2 ), thaw (60°C) and vortex shaking. The alignment of bicelles was 'flipped' with the normal of the bilayer parallel to the magnetic field by adding the lanthanide salt YbCl 3 at a concentration of 2.5 mM 31,35 for the binary systems D14PC/14PC and D16PC/14PC.…”

“…All solution 31 P NMR experiments were carried out on an Avance III HD 600 MHz spectrometer (Bruker, Milton, ON, Canada) equipped with a 5-mm double-resonance probe. A single-pulse experiment was employed with a π/2 pulse of 15 µs, a recycle delay of 5 s, and an acquisition time of 1 s with broadband proton continuous wave decoupling at a field strength of 5 kHz.…”

“…Specifically, dodecyl-(12PC), tetradecyl-(14PC) and hexadecyl (16PC) phosphocholines were employed. The detergent 12PC is often used to solubilize membrane proteins, and has previously been used with D14PC to prepare isotropic and magnetically-oriented bicelles 30,31,33 . When compared to 12PC, 14PC micelles have shown their ability to maintain the activity of dialkylglycerol kinase (DAGK) due to their better match with the hydrophobic span of the transmembrane domain of this protein 34 .…”

“…Triton X-100 detergent demonstrates a better magnetic alignment and stability of membrane proteins in the bilayer 28 , while addition of cholesterol, cholesterol 3-sulfate and/or hexadecyl-trimethylammonium bromide (CTAB) stabilizes the bilayer and increases the temperature range at which bicelles form 29 . To obtain bicelles at low concentrations and more suitable for solution NMR, detergents with low critical micelle concentration (CMC) were used such as dodecyl-PC (12PC) and several cyclohexyl-1-butyl-PC [30][31][32] .…”