Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography

Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.     

The vesicle-to-micelle transition of phosphatidylcholine vesicles induced by nonionic detergents: effects of sodium chloride, sucrose and urea

The vesicle-to-micelle transition of egg phosphatidylcholine LUVs induced by octylglucoside was studied in buffers with 0-4 M sodium chloride, sucrose or urea. We used both light scattering and fluorescent probes to follow the lipid-detergent complexes in these buffers. The vesicle-to-micelle transition process was fundamentally the same in each solute. However, the detergent-to-lipid ratio required for micelle formation shifted in ways that depended on the aqueous solute. The partitioning of octylglucoside between the vesicles and the aqueous phase was primarily determined by the change in its critical micelle concentration (cmc) induced by each solute. Specifically, the cmc decreased in high salt and sucrose buffers but increased in high concentrations of urea. Cmc for two additional nonionic detergents, decyl- and dodecyl-maltoside, and three zwittergents (3-12, 3-14 and 3-16) were determined as a function of concentration for each of the solutes. In all cases NaCl and sucrose decreased the solubility of the detergents, whereas urea increased their solubilities. The effects clearly depended on acyl chain length in urea-containing solutions, but this dependence was less clear with increasing NaCl and sucrose concentrations. The contributions of these solutes to solubility and to interfacial interactions in the bilayers, pure and mixed micelles are considered.     

The physicochemical state of protoporphyrin IX in aqueous solution investigated by fluorescence and light scattering

Fluorescence spectros copy and light scattering have been used to investigate the physicochemical behaviour of protoporphyrin IX in aqueous solutions. In the alkaline range large micelles are formed with a hydrodynamic radius of 130 nm and a molecular mass of 5.0 x 10(7) Da. The micelles are fluorescent with an emission maximum at 620 nm. A pH lowering caused quenching of the micelle fluorescence. On a collision encounter these micelles will disintegrate and they are reformed by nucleation of collision fragments. From measurements of the fluorescence intensity of the micelles versus total concentration an equilibrium constant of 4.0 x 10(6) M(-1) was found for this collision-nucleation process. In the pH range between 6 and 3 another micelle type of twice the size of those in the alkaline range was stable with respect to the solute. These micelles have free base porphyrin fluorescence with an emission maximum at 634 nm. A lowering of the pH below unity causes disintegration of these micelles and monomer fluorescence from the protoporphyrin dication was observed.     

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.     

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.     

Ultrastructure of human erythrocyte GLUT1

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.     

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.     

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.     

Fluidity of detergent micelles plays an important role in muscarinic receptor solubilization

In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active conformation of the solubilized muscarinic receptor.     

Characterization of mixed micelles of phospholipids of various classes and a synthetic,homogeneous analogue of the nonionic detergent triton X-100 containing nine oxyethylene groups

The synthesis and high-pressure liquid chromatographic purification of the homogeneous nonionic surfactant $\text{p-}\text{(1,1,3,3-tetramethylbutyl)phenoxynonaoxyethylene}$ glycol (OPE-9) in quantities suitable for membrane solubilization studies is reported. Micelles of OPE-9 and mixed micelles of OPE-9 with dimyristoyl and dipalmitoyl phosphatidylcholine as well as phosphatidylserine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, and palmitic acid were characterized by column chromatography on 6% agarose. It was found that at 28°C OPE-9 micelles have a Stokes' radius of 32 Å, giving a molecular weight for a spherical micelle of about half that of micelles of the polydisperse nonionic surfactant Triton X-100 under the same conditions. The micelle size is temperature dependent: at 40°C the OPE-9 micelles have a Stokes' radius of 44 Å, giving a molecular weight for a spherical micelle of about twice that of the OPE-9 micelles at 28°C. The size of the mixed micelles varies linearly (as measured by $\text{K}{}_{\mathrm{<mtext>av</mtext>}}$) with the mole fraction of phospholipid. The mixed micelle size was found to be relatively independent of the absolute concentration of surfactant over a four-fold range if the mole fraction of phospholipid is kept constant. The usefulness of the OPE-9/phospholipid mixed micelle system for lipolytic enzyme substrates and membrane-related studies is considered.     

The Fluidity of Phosphocholine and Maltoside Micelles and the Effect of CHAPS

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.e., fluidity). The nitroxide probe rotational dynamics in longer chain detergents is more restricted than in shorter chain detergents, and maltoside micelles are more restricted than phosphocholine micelles. Furthermore, the micelle microviscosity can be modulated with mixtures, as demonstrated with mixtures of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate with n-dodecylphosphocholine, n-tetradecylphosphocholine, n-decyl-β-D-maltoside, or n-dodecyl-β-D-maltoside. These results indicate that observed differences in membrane protein stability in these detergents could be due to fluidity in addition to the already determined structural differences.     

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。     

Structure of bovine casein oligomers

We have characterized the size, molecular weight, and composition of the oligomeric particles produced by dialysis of bovine casein micelles against solutions lacking calcium ion. The particles were stabilized against further dissociation after dialysis by glutaraldehyde fixation. The progress of the dissociation was monitored by Biogel A-15 gel permeation chromatography, sucrose density gradient ultracentrifugation, inelastic laser light scattering, sedimentation velocity and equilibrium, and urea starch gel electrophoresis. The casein oligomers isolated after dialysis against either 0.5 m NaCl/SMUF A/azide or calcium-free SMUF/ azide have a hydrodynamic radius of about 5.5 nm and a molecular weight of about 95,000 corresponding to roughly four casein monomers (SMUF, simulated milk ultrafiltrate). The oligomers are highly hydrated and contain one-third of the calcium ion found in native micelles. During the course of dialysis, the micelles gradually break down into a broad distribution of intermediatesized particles and then into the oligomers described above.     

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.     

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.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 degrees C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 degrees C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Small-angle neutron scattering study of the association between porcine pancreatic colipase and taurodeoxycholate micelles

Small-angle neutron scattering studies have shown the association of porcine colipase with bile salts micelles to be a lateral one. The molecular structure parameters of the individual components were determined first. A radius of gyration of 13.9 Å is found for colipase, which implies a non-spherical shape for this molecule. The size of taurodeoxycholate micelles is controlled by the ionic strength of the solution. In 0.15 m-NaCl their volume is comparable to that of colipase; they are elongated with an axial ratio of about 2. At higher ionic strengths the elongation of the micelles increases.In 0.15 m-NaCl the complex is found to be an association of one colipase molecule with a volume of detergent corresponding to that of one free micelle. The contrast variation study of the radius of gyration shows that in the complex the centre of masses of the protein and of the detergent are well-separated: a distance between 29 and 45 Å has been estimated. The value of the radius of gyration of the complex at high contrast, and the agreement between the contrast variation analysis and a straightforward application of the parallel axes theorem indicate that the complex is formed by the juxtaposition of the protein and a preformed micelle, which has approximately the same size and shape as a free micelle. There is only one localized surface contact between the protein and the micelle, which implies that colipase possesses a relatively well-defined binding site.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 °C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 °C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution.Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.