Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

Abstract

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family display favorable behavior relative to conventional detergents, as tested on multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

“…This last result suggests that consequences of incubating LeuT with CMI are more pronounced Binding of substrate to LeuT F253A H. Wang & E. Gouaux scientific report in C 12 M than in MNG-3, and that MNG-3 is a superior detergent for LeuT ligand binding studies. Our fluorescence-detection sizeexclusion chromatography experiment (supplementary Fig S3 online) further demonstrates that 1 mM CMI can diminish the protein concentrations to a greater extent in C 12 M than in MNG-3, an observation that is also consistent with the notion MNG-3 better stabilizes apo LeuT [24]. The mechanism by which CMI affects the solubility of LeuT is presently unclear.…”

“…The K d values for the WT transporter are similar to those measured with the protein in C 12 M, whereas we observed an increase in K d to 406.7±33.5 nM for the F253A mutant in MNG-3 detergent (supplementary Table I online). Most importantly, we found that in MNG-3 the B max values did not decrease as much as in C 12 M on preincubation of the WT and mutant proteins with CMI (supplementary Fig S2 online), consistent with the notion that MNG-3 better stabilizes both the WT and F253A mutant of apo LeuT [24].…”

“…We therefore carried out the same leucine saturation binding experiments as described above using the new detergent MNG-3, which has previously been shown to better stabilize LeuT in comparison to C 12 M [24]. For both the WT and the F253A mutant, we measured K d values in the absence and presence of CMI (Fig 1).…”

Structure of the Full-Length TRPV2 Channel by Cryo-EM

“…Conversely, the TM domain of the proteins was stabilized in different manners during the purification process. While apo-TRPV1 and ligand-bound TRPV1 were stabilized in A8-35 amphipol 5,6,29-31 , TRPV2 was purified using the maltose neopentyl glycol (MNG) class of detergents 16,31,32 . This difference in the biochemical preparations may have enabled the TRPV preparations to sample different conformational states, resulting in the observed divergence in the channel architectures [29][30][31] .…”

Manipulation des récepteurs couplés aux protéines G

“…Dans la première catégorie de molécules se trouvent notamment les MNG (maltose-néopentylglycol) [33]. Ces composés sont des déter-gents originaux qui ont pour principale caractéristique la présence d'un carbone quaternaire central qui restreint la flexibilité conformationnelle de la molécule.…”