Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.     

Membrane proteins, lipids and detergents: not just a soap opera

Studying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will concentrate on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant molecular properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystallization. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the 'equilibrium partition model', the dependence of the 'solubilizing detergent concentration' on the lipid concentration intersects with the lipid axis at -1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical ('solubilizing') effective ratio depends upon the nature of detergent less than any of these two factors.     

去污剂的性质及其在膜蛋白结构生物学中的应用

膜蛋白在诸多生物过程,如呼吸作用、光合作用、信号识别和分子转运等方面发挥着重要作用,近年来,去污剂的快速发展,在一定程度上极大地推动了膜蛋白研究的进展。去污剂广泛应用于膜蛋白的提取、增溶、纯化、理化性质及结构研究,然而如何选择合适的去污剂往往是一项复杂的任务。本文从以下两个方面入手系统地描述了去污剂的重要理化性质及其在膜蛋白结构功能研究中的应用,（1）去污剂结构及其对去污剂性质和水溶性的影响,去污剂形成胶束的条件及影响去污剂胶束形成的其他因素。希望这些关于去污剂的基本性质和参数的介绍,可以为相关科研工作者选用去污剂提供一个理论依据。（2）去污剂抽提膜蛋白的流程和注意细节,去污剂对膜蛋白纯化时分子量测定的影响,膜蛋白研究中去污剂的置换与去除,膜蛋白结构、功能研究案例归纳。希望这些应用细节、课题研究,可以为相关科研工作者研究膜蛋白结构功能时提供一个经验借鉴。     

去污剂的性质及其在膜蛋白结构生物学中的应用

膜蛋白在诸多生物过程,如呼吸作用、光合作用、信号识别和分子转运等方面发挥着重要作用,近年来,去污剂的快速发展,在一定程度上极大地推动了膜蛋白研究的进展。去污剂广泛应用于膜蛋白的提取、增溶、纯化、理化性质及结构研究,然而如何选择合适的去污剂往往是一项复杂的任务。本文从以下两个方面入手系统地描述了去污剂的重要理化性质及其在膜蛋白结构功能研究中的应用,（1）去污剂结构及其对去污剂性质和水溶性的影响,去污剂形成胶束的条件及影响去污剂胶束形成的其他因素。希望这些关于去污剂的基本性质和参数的介绍,可以为相关科研工作者选用去污剂提供一个理论依据。（2）去污剂抽提膜蛋白的流程和注意细节,去污剂对膜蛋白纯化时分子量测定的影响,膜蛋白研究中去污剂的置换与去除,膜蛋白结构、功能研究案例归纳。希望这些应用细节、课题研究,可以为相关科研工作者研究膜蛋白结构功能时提供一个经验借鉴。     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, R_e. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the ‘equilibrium partition model’, the dependence of the ‘solubilizing detergent concentration’ on the lipid concentration intersects with the lipid axis at −1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical (‘solubilizing’) effective ratio depends upon the nature of detergent less than any of these two factors.     

Evidence for a tubulin-containing lipid-protein structural complex in ciliary membranes

The proteins and lipids of the scallop gill ciliary membrane may be reassociated through several cycles of detergent solubilization, detergent removal, and freeze-thaw, without significant change in overall protein composition. Membrane proteins and lipids reassociate to form vesicles of uniform, discrete density classes under a variety of reassociation conditions involving detergent removal and concentration. Freed of the solubilizing detergent during equilibrium centrifugation, a protein-lipid complex equilibrates to a position on a sucrose density gradient characteristic of the original membrane density. When axonemal tubulin is solubilized by dialysis, mixed with 2:1 lecithin/cholesterol dissolved in Nonidet P-40, freed of detergent, and reconstituted by freeze-thaw, vesicles of a density essentially equal to pure lipid result. If the lipid fraction is derived through chloroform-methanol extraction of natural ciliary membranes, a moderate increase in density occurs upon reconstitution, but the protein is adsorbed and most is removed by a simple low ionic strength wash, in contrast to vesicles reconstituted from membrane proteins where even high salt extraction causes no loss of protein. The proteins of the ciliary membrane dissolve with constant composition, regardless of the type, concentration, or efficiency of detergent. Analytical ultracentrifugation demonstrates that monodisperse mixed micelles form at high detergent concentrations, but that membranes are dispersed to large sedimentable aggregates by Nonidet P-40 even at several times the critical micelle concentration, which suggests reasons for the efficacy of certain detergent for the production of ATP-reactivatable cell models. In extracts freed of detergent, structured polydisperse particles, but not membrane vesicles, are seen in negative staining; vesicles form upon concentration of the extract. Membrane tubulin is not in a form that will freely undergo electrophoresis, even in the presence of detergent above the critical micelle concentration. All chromatographic attempts to separate membrane tubulin from other membrane proteins have failed; lipid and protein are excluded together by gel filtration in the presence of high concentrations of detergent. These observations support the idea that a relatively stable lipid-protein complex exists in the ciliary membrane and that in this complex membrane tubulin is tightly associated with lipids and with a number of other proteins.     

Detergent organisation in solutions and in crystals of membrane proteins

The use of neutron scattering in studying the organisation of detergents in pure micelles, in protein/detergent mixed micelles and in crystals of membrane proteins, is reviewed. Small angle scattering has been used to study the size, shape and composition of pure and mixed protein/detergent micelles as well as the effects of adding small amphiphiles. The technique of contrast variation applied to single crystals is described and its application to the determination of the organization of detergent in single crystals of membrane proteins is discussed. A better understanding of protein/detergent interactions should help in producing crystals of membrane proteins more easily as well as clues to the nature of protein/lipid interactions in vivo.     

Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase.

As chaotropic salts are generally believed to affect water structure in a manner which increases lipophilicity of water, they may seem to be capable of substituting for detergents in the solubilization of particulate enzyme. Although solubilization either by detergents or by chaotropic salts has been demonstrated with several membrane proteins, the effects these agents have on the properties and activity of an enzyme may be quite different. This is illustrated by the effects on mammalian mitochondrial dihydroorotate dehydrogenase. Stability of the solubilized enzymic activity is dependnet on the presence of a detergent and maximum enzymic activity is observed at the critical micelle concentration of the detergent. Addition of low concentrations of various anions of the chaotropic series further enhances activity while higher concentrations of these anions, although increasing solubility of the enzyme, irreversibly inhibit catalysis.     

Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.     

Detergent Properties Influence the Stability of the Glycophorin A Transmembrane Helix Dimer in Lysophosphatidylcholine Micelles

Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important.     

Sucrose monoester micelles size determined by Fluorescence Correlation Spectroscopy (FCS)

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R(h). As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured.     

Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers

Abstract

Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.     

The interaction of detergents with bilayer lipid membranes

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025–0.15 mM; dodecyl sulphate 0.82 in the range 0.01–1 mM; sodium deoxycholate 1.03 in the range 0.01–1 mM and sodium cholate 1.37 in the range 0.1–10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.     

A sensitive method for staining proteins transferred to nitrocellulose sheets

A simple physical method to determine the monomer concentration of detergents below as well as above the critical micelle concentration based on the bubble-pressure measurement is described. Aggregated surfactant molecules (micelles) and phospholipid vesicles if present in the sample will not disturb the measurements. Three applications of the method relevant to the preparation of liposomes are shown: (i) measurements of critical micelle concentrations, (ii) evaluation of the affinity constant of the interaction of detergents with liposomal membranes, and (iii) monitoring of residual detergent in liposome preparations during dialysis or after gel chromatography of mixed micelle-derived liposomes. It was found that the efficiency of detergents to produce liposomes during their removal depends on their critical micelle concentrations as well as on their affinity to liposomal membranes.     

A gas chromatography–mass spectrometry method to monitor detergents removal from a membrane protein sample

In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.     

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.     

Negative staining of integral membrane proteins

This review describes aspects of negative staining of isolated integral membrane proteins. Detergents play a central role in the isolation of membrane proteins and also in their solubility in aqueous solutions. Specimens of mixed micelles of membrane proteins and nonionic detergents can be easily prepared as long as the detergent concentration remains above the critical micellar concentration. Membrane proteins involved in the process of photosynthesis have been taken as examples to illustrate their interaction with different detergents. Upon negative staining, mixed micelles of membrane proteins and detergents show characteristic top and side view projections. On their sides, mixed micelles can easily aggregate into strings.     

Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.