Quantification of Detergents Complexed with Membrane Proteins

Abstract

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.

An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization

“…It has CMC value of 0.01%/0.2 mM, with the micelle size of 60 to 90 kDa (temperature-dependent) [63,64]. LMNG has a low CMC value (0.001%/0.01 mM [33]), with the size of micelle of Mw ~ 91 kDa (this work), which is in contrast with the recent result of ~ 393 kDa obtained with MALDI-TOF MS [19]. It has been reported to be efficient both at protein extraction and stabilization of several delicate membrane proteins [37,61,62].…”

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins

“…[5] Detergents,c ommonly used to extract MPs,are amphipathic molecules with apolar head and ahydrophobic counterpart. They form complexes with MPs, [6] preventing irreversible aggregation in aqueous solution. However,b eing more exchangeable than lipids, [7] detergents tend to expose hydrophobic patches of the membrane domain of MPs [6] to water, which contributes to reducing their stability and promotes aggregation.…”

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction, Overstabilization, and Crystallization of Membrane Proteins

“…[4] Am ajor challenge encountered with MPs during extraction, purification, and structural experiments is the change from the native lipophilic environment to an aqueous one,which can lead to the loss of the active conformation. They form complexes with MPs, [6] preventing irreversible aggregation in aqueous solution. They form complexes with MPs, [6] preventing irreversible aggregation in aqueous solution.…”

“…[5] Detergents,c ommonly used to extract MPs,are amphipathic molecules with apolar head and ahydrophobic counterpart. However,b eing more exchangeable than lipids, [7] detergents tend to expose hydrophobic patches of the membrane domain of MPs [6] to water, which contributes to reducing their stability and promotes aggregation. However,b eing more exchangeable than lipids, [7] detergents tend to expose hydrophobic patches of the membrane domain of MPs [6] to water, which contributes to reducing their stability and promotes aggregation.…”

“…Black, gray,and white bars correspond to the detergent concentration at which BmrA is either fully,p artially,o rnot solubilized, as deduced from Figure S6. We determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry,a sw er ecently set up, [6] that most of the DDM had indeed been exchanged by this procedure (not shown). Figure S9.…”