Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins

Abstract

2Laurylmaltose neopentylglycol (LMNG) bears two linked hydrophobic chains of equal length 3 and two hydrophilic maltoside groups. It arouses a strong interest in the field of membrane 4 protein biochemistry, since it was shown to efficiently solubilize and stabilize membrane 5 proteins often better than the commonly used dodecylmaltopyranoside (DDM), and to allow 6 structure determination of some challenging membrane proteins. However, LMNG was 7 described to form large micelles, which could be unfavorable for structural purposes. We thus 8 investigated its auto-assemblies and the association state of different membrane proteins 9 solubilized in LMNG by analytical ultracentrifugation, size exclusion chromatography 10 coupled to light scattering, centrifugation on sucrose gradient and/or small angle scattering. 11 At high concentrations (in the mM range) LMNG forms long rods, and it stabilized the 12 membrane proteins investigated herein, i.e. a bacterial multidrug transporter, BmrA; a 13 prokaryotic analogous of the eukaryotic NADPH oxidases, SpNOX; an E. coli outer 14 membrane transporter, FhuA; and the halobacterial bacteriorhodopsin, bR. BmrA, in the Apo 15 and the vanadate-inhibited forms showed reduced kinetics of limited proteolysis in LMNG 16 compared to DDM. Both SpNOX and BmrA display an increased specific activity in LMNG 17 compared to DDM. The four proteins form LMNG complexes with their usual quaternary 18 structure and with usual amount of bound detergent. No heterogeneous complexes related to 19 the large micelle size of LMNG alone were observed. In conditions where LMNG form 20 assemblies of large size, FhuA crystals diffracting to 4.0 Å were obtained by vapor diffusion. 21 LMNG large micelle size thus does not preclude membrane protein homogeneity and 22 crystallization.23 24 29Detergents are essential for membrane protein solubilization and purification steps, as well as 30 functional and structural studies. However, they often lead to membrane protein inactivation. 31One of the mechanisms leading to inactivation (for a recent review, see [2]) is the dissociation 32 of subunits, loss of essential lipids or other hydrophobic co-factors. As an example, 33 cytochrome b 6 f inactivation and monomerization was related to lipid loss [3]. Detergent 34 dynamics, i.e. detergent exchange between the free monomers, or between micelles, and 35 bound detergent at the surface of the transmembrane protein surface, could trigger transient 36 exposure of this hydrophobic surface leading to irreversible protein aggregation. Detergent 37 binding can also alter protein structure: molecular dynamics simulation indeed showed, for a 38 thermostable mutant of the adenosine receptor, that the harsh octylglucoside detergent 39 molecules intercalate between trans-membrane helices, moving them apart in the 200 ns 40 simulation [4]. The structure and dynamics of different -helical membrane proteins 41 solubilized in alkyl phosphocholines appear deeply altered [5]. For example, NMR, molecular 42 dynamics simulations, and fu...

Mass Photometry of Membrane Proteins

“…We began with the detergent lauryl maltose-neopentyl glycol (LMNG) due to its popularity in structural studies, based on enhanced stability of membrane proteins solubilized in this manner. 11 In detergent micelles, using an abundance of detergent above the critical micelle concentration (CMC) aids in solubilizing hydrophobic, transmembrane regions of the IMP, thereby maintaining solubility. To assess the signal generated by detergent micelles in MP, we compared LMNG at 2.53 CMC (0.0025% w/v) in buffer (50 mM MOPS, 20 mM NaCl, pH 7.5) with a buffer blank ( Figure 1B).…”

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

“…The data were processed using the protein conjugate analysis program within the ASTRA software (Wyatt Technology). A dn/dc value of 0.14 ml/g was used for LMNG (41,42). FtsH samples were run over a Superose 6 Increase 10/300 GL column in 20 mM Tris-HCl pH 8.0; 150 mM NaCl; 0.01 % LMNG at room temperature.…”

Mass Photometry of Membrane Proteins

“…To assess the effect of an abundance of detergent micelles, we compared LMNG at 2.5x CMC (0.0025% w/v) in buffer (50 mM MOPS, 20 mM NaCl, pH 7.5) compared to a buffer blank (Figure 1b). We observed considerable non-specific detergent interactions (Figure 1c and Supplementary Movie 2), due to the effective concentration of LMNG micelles at 2.5x CMC (25 µM) 11 being an order of magnitude larger than the current optimal concentration range for MP (high pM to mid nM). This caused surface saturation and rapid binding and unbinding on the glass interface, effectively increasing the imaging background, reducing mass resolution and raising the lower detection limit.…”

“…To assess the extent to which these could hamper MP measurements, and to help interpret downstream measurements of complex systems, we first studied commonly used membrane mimetic systems in the absence of protein. We began with the detergent lauryl maltose-neopentyl glycol (LMNG) due to its popularity in structural studies based on enhanced stability of membrane proteins solubilised in this manner 11 .…”