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.     

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

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.     

Non-ionic hybrid detergents for protein delipidation

Non-ionic detergents are important tools for the investigation of interactions between membrane proteins and lipid membranes. Recent studies led to the question as to whether the ability to capture protein-lipid interactions depends on the properties of detergents or their concentration in purification buffers. To address this question, we present the synthesis of an asymmetric, hybrid detergent that combines the head groups of detergents with opposing delipidating properties. We discuss detergent properties and protein purification outcomes to reveal whether the properties of detergent micelles or the detergent concentration in purification buffers drive membrane protein delipidation. We anticipate that our findings will enable the development of rationally design detergents for future applications in membrane protein research.     

Solubilization of phospholipids by detergents. Structural and kinetic aspects

Most amphiphiles in biological membranes including phospholipids, steroids, and membrane proteins are insoluble amphiphiles and would form liquid crystals or insoluble precipitates alone in aqueous media. Detergents are soluble amphiphiles and above a critical concentration and temperature form micelles of various sizes and shapes. Much of the recent progress in studying the insoluble amphiphiles is due to the formation of thermodynamically stable isotropic solutions of these compounds in the presence of detergents. This process, which is commonly denoted as "solubilization,' involves transformation of lamellar structures into mixed micelles. The information available to date on the solubilization of phospholipids, which constitute the lipid skeleton of biomembranes, by the common detergents is discussed in this review, both with respect to the kinetics of this process and the structure of the various phospholipid-detergent mixed micelles formed. It is hoped that this discussion will lead to somewhat more useful, although still necessarily fairly empirical, approaches to the solubilization of phospholipids by detergents.     

Lipopeptide detergents designed for the structural study of membrane proteins

The structural study of membrane proteins requires detergents that can effectively mimic lipid bilayers, and the choice of detergent is often a compromise between detergents that promote protein stability and detergents that form small micelles. We describe lipopeptide detergents (LPDs), a new class of amphiphile consisting of a peptide scaffold that supports two alkyl chains, one anchored to each end of an alpha-helix. The goal was to design a molecule that could self-assemble into a cylindrical micelle with a rigid outer hydrophilic shell surrounding an inner lipidic core. Consistent with this design, LPDs self-assemble into small micelles, can disperse phospholipid membranes, and are gentle, nondenaturing detergents that preserve the structure of the membrane proteins in solution for extended periods of time. The LPD design allows for a membrane-like packing of the alkyl chains in the core of the molecular assemblies, possibly explaining their superior properties relative to traditional detergents in stabilizing membrane protein structures.     

Sequence-Specific Dimerization of a Transmembrane Helix in Amphipol A8-35

As traditional detergents might destabilize or even denature membrane proteins, amphiphilic polymers have moved into the focus of membrane-protein research in recent years. Thus far, Amphipols are the best studied amphiphilic copolymers, having a hydrophilic backbone with short hydrophobic chains. However, since stabilizing as well as destabilizing effects of the Amphipol belt on the structure of membrane proteins have been described, we systematically analyze the impact of the most commonly used Amphipol A8-35 on the structure and stability of a well-defined transmembrane protein model, the glycophorin A transmembrane helix dimer. Amphipols are not able to directly extract proteins from their native membranes, and detergents are typically replaced by Amphipols only after protein extraction from membranes. As Amphipols form mixed micelles with detergents, a better understanding of Amphipol-detergent interactions is required. Therefore, we analyze the interaction of A8-35 with the anionic detergent sodium dodecyl sulfate and describe the impact of the mixed-micelle-like system on the stability of a transmembrane helix dimer. As A8-35 may highly stabilize and thereby rigidify a transmembrane protein structure, modest destabilization by controlled addition of detergents and formation of mixed micellar systems might be helpful to preserve the function of a membrane protein in Amphipol environments.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.     

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.     

Hemifluorinated surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions?

The instability of membrane proteins in detergent solution can generally be traced to the dissociating character of detergents and often correlates with delipidation. We examine here the possibility of substituting detergents, after membrane proteins have been solubilized, with non-detergent surfactants whose hydrophobic moiety contains a perfluorinated region that makes it lipophobic. In order to improve its affinity for the protein surface, the fluorinated chain is terminated by an ethyl group. Test proteins included bacteriorhodopsin, the cytochrome b(6)f complex, and the transmembrane region of the bacterial outer membrane protein OmpA. All three proteins were purified using classical detergents and transferred into solutions of C(2)H(5)C(6)F(12)C(2)H(4)-S-poly-Tris-(hydroxymethyl)aminomethane (HF-TAC). Transfer to HF-TAC maintained the native state of the proteins and prevented their precipitation. Provided the concentration of HF-TAC was high enough, HF-TAC/membrane protein complexes ran as single bands upon centrifugation in sucrose gradients. Bacteriorhodopsin and the cytochrome b(6)f complex, both of which are detergent-sensitive, exhibited increased biochemical stability upon extended storage in the presence of a high concentration of HF-TAC as compared to detergent micelles. The stabilization of cytochrome b(6)f is at least partly due to a better retention of protein-bound lipids.     

Reconstitution of a protein into lipid vesicles using natural detergents

A method is described for reconstitution of a protein into lipid vesicles using one of the natural detergents lysophosphatidylcholine or lysophosphatidic acid. The intestinal microvillus enzyme, aminopeptidase N (EC 3.4.11.2) is incorporated into lipid vesicles prepared from a total lipid extract of the microvillus membrane. The method is based on fusion of aminopeptidase-lysophospholipid micelles with liposomes prepared by sonication. The incorporation of the protein into the lipid bilayer is analyzed by gel permeation chromatography and sucrose density gradient centrifugation. The coincidence of the protein and lipid profiles is used to evaluate protein incorporation. The incorporation is visualized by electron microscopy with negative staining. The method has the advantage of using natural detergents, lysophospholipids, which are minor but natural constituents of biological membranes. The method could be of value as a tool in studies of mechanisms of insertion of newly synthesized proteins into biological membranes.     

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

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。     

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.     

Conformations of peptides derived from myelin-specific proteins in membrane-mimetic conditions probed by synchrotron radiation CD spectroscopy

Myelin is a tightly packed membrane multilayer in the nervous system, which harbours a specific set of quantitatively major proteins. All these proteins interact with the lipid bilayer, being either peripheral or integral membrane proteins. In this study, we examined the conformational properties of peptides from the myelin proteins P0, CNPase, MOBP, P2 and MOG, using trifluoroethanol and micelles of different detergents as membrane-like mimics. The peptides showed significant differences in their folding under the employed conditions, as evidenced by synchrotron radiation circular dichroism spectroscopy. Our experiments provide new structural information on the interactions between myelin proteins and membranes, using a simplified model system of synthetic peptides and micelles.     

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.     

Phase Separation of Biomolecules in Polyoxyethylene Glycol Nonionic Detergents

Abstract

The advantage of aqueous two-phase systems based on polyoxyethylene detergents over other liquid-liquid two-phase systems lies in their capacity to fractionate membrane proteins simply by heating the solution over a biocompatible range of temperatures (20 to 37°C). This permits the peripheral membrane proteins to be effectively separated from the integral membrane proteins, which remain in the detergent-rich phase due to the interaction of their hydrophobic domains with detergent micelles. Since the first reports of this special characteristic of polyoxyethylene glycol detergents in 1981, numerous reports have consolidated this procedure as a fundamental technique in membrane biochemistry and molecular biology. As examples of their use in these two fields, this review summarizes the studies carried out on the topology, diversity, and anomalous behavior of transmembrane proteins on the distribution of glycosyl-phosphatidylinositol-anchored membrane proteins, and on a mechanism to describe the pH-induced translocation of viruses, bacterial endotoxins, and soluble cytoplasmic proteins related to membrane fusion.

In addition, the phase separation capacity of these polyoxyethylene glycol detergents has been used to develop quick fractionation methods with high recoveries, on both a micro- and macroscale, and to speed up or increase the efficiency of bioanalytical assays.     

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.     

Deuterated detergents for structural and functional studies of membrane proteins: Properties,chemical synthesis and applications

Abstract

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.