Protein-surfactant interactions: a tale of many states

The scientific study of protein surfactant interactions goes back more than a century, and has been put to practical uses in everything from the estimation of protein molecular weights to efficient washing powder enzymes and products for personal hygiene. After a burst of activity in the late 1960s and early 1970s that established the general principles of how charged surfactants bind to and denature proteins, the field has kept a relatively low profile until the last decade. Within this period there has been a maturation of techniques for more accurate and sophisticated analyses of protein-surfactant complexes such as calorimetry and small angle scattering techniques. In this review I provide an overview of different useful approaches to study these complexes and identify eight different issues which define central concepts in the field. (1) Are proteins denatured by monomeric surfactant molecules, micelles or both? (2) How does unfolding of proteins in surfactant compare with "proper" unfolding in chemical denaturants? Recent work has highlighted the role of shared micelles, rather than monomers, below the critical micelle concentration (cmc) in promoting both protein denaturation and formation of higher order structures. Kinetic studies have extended the experimentally accessible range of surfactant concentrations to far above the cmc, revealing numerous different modes of denaturation by ionic surfactants below and above the cmc which reflect micellar properties as much as protein unfolding pathways. Uncharged surfactants follow a completely different denaturation strategy involving synergy between monomers and micelles. The high affinity of charged surfactants for proteins means that unfolding pathways are generally different in surfactants versus chemical denaturants, although there are common traits. Other issues are as follows: (3) Are there non-denaturing roles for SDS? (4) How reversible is unfolding in SDS? (5) How do solvent conditions affect the way in which surfactants denature proteins? The last three issues compare SDS with "proper" membranes. (6) Do anionic surfactants such as SDS mimic biological membranes? (7) How do mixed micelles interact with globular proteins? (8) How can mixed micelles be used to measure the stability of membrane proteins? The growing efforts to understand the unique features of membrane proteins have encouraged the development of mixed micelles to study the equilibria and kinetics of this class of proteins, and traits which unite globular and membrane proteins have also emerged. These issues emphasise the amazing power of surfactants to both extend the protein conformational landscape and at the same time provide convenient and reversible short-cuts between the native and denatured state for otherwise obdurate membrane proteins.     

Fluorinated and hemifluorinated surfactants derived from maltose: synthesis and application to handling membrane proteins in aqueous solution

Two novel fluorinated surfactants have been obtained by grafting by radical reaction either a fluorocarbon or an ethyl end-capped fluorocarbon chain onto the double bond of beta-D-allyl maltose. The two compounds thus obtained form polydisperse aggregates in water. They can keep membrane proteins water-soluble, but the protein/surfactant complexes are polydisperse, which affects neither the native state nor the stability of the proteins.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.     

Structural characteristics of extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 core antenna complexes CP43 and CP47

The structural characteristics of the extra-membrane domains and guanidine hydrochloride-induced denaturation of photosystem 2 (PS2) core antenna complexes CP43 and CP47 were investigated using fluorescence emission and circular dichroism (CD) spectra. The extra-membrane domains of CP43 and CP47 possessed a certain degree of secondary and tertiary structure and not a complete random coil conformation. The tertiary structure and the chlorophyll (Chl) a microenvironment of CP47 were more sensitive to guanidine hydrochloride (GuHCl) than that of CP43. Changes in energy transfer from β-carotene to Chl a corresponded well to changes in the tertiary structure while their correlation with changes in the secondary structure was rather poor. Unlike most of water-soluble proteins, both CP43 and CP47 are partly resistant to denaturation induced by guanidine hydrochloride (GuHCl); the denaturation of CP43 or CP47 is not a two-state process. Those features most probably reflect their character as intrinsic membrane proteins.     

Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.     

Bioconjugated phospholipid polymer biointerface for enzyme-linked immunosorbent assay

This study aimed to develop a sensitive and reliable immunoassay by applying a highly functional phospholipid polymer biointerface. We synthesized a phospholipid polymer--poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). MEONP contains active ester groups on the side chains for immobilization of antibodies via oxyethylene. PMBN with different compositions and oxyethylene chain lengths were synthesized; their effects on nonspecific and specific values in the immunoassay were evaluated. MPC units reduce the background by preventing nonspecific protein adsorption. MEONP units could conjugate antibodies and enhance the specific signal. The specific signal was independent of the oxyethylene chain length, but long oxyethylene chains increased the background. Specific signals corresponding to the antigen were observed with the PMBN coating, and a liner standard curve was obtained. The PMBN-coated surface maintained residual activity after long-term storage. This surface affords a low background without requiring blocking treatment and is suitable for immobilized antibodies.     

Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.     

A Microplate Assay for DNA Damage Determination (Fast Micromethod)in Cell Suspensions and Solid Tissues

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 μg tissue per well), or in environmental samples for genotoxicity assessment.     

Forces and factors that contribute to the structural stability of membrane proteins

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.     

The starch granule associated proteomes of commercially purified starch reference materials from rice and maize

Commercially available reference materials are integral components of many experimental protocols, as it is critical to compare one's results to those derived from well-characterized standards. Most reference materials are well defined, with all their components being cataloged. However, certain reference materials, such as commercially prepared starch samples, can have undefined components, potentially limiting their usefulness as standards. The proteome of commercially prepared starch has not been documented, and to that end, we initiated a mass spectrometry-based survey of the proteins associated with starch granules in commercially prepared rice and maize starch samples. We performed direct trypsin treatments of starch samples and sequenced both the water-soluble peptides liberated into the aqueous supernatant and the peptides released from the starch granule surface by isopropanol solvent washing. We discovered that the majority of proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. We also documented proteins that are markers for seed maturity and for starch mobilization.     

A microplate assay for DNA damage determination (fast micromethod).

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 microg tissue per well), or in environmental samples for genotoxicity assessment.     

Conformational stability of spectrin and fodrin

The conformational stability of erythrocyte spectrin and brain spectrin-like protein (fodrin) has been studied by circular dichroism. In agreement with previous reports the circular dichroism spectra of both proteins in the peptide region were almost identical. The essential differences, on the other hand, were found in the near u.v. range, most probably due to differences in the conformation of intrachain disulphide bonds. Heat denaturation curves, relating to the level of secondary structure (ellipticity at 221 nm) showed that fodrin is more stable than spectrin: curves of reversible as well as irreversible denaturation are shifted to higher temperatures and also the amount of alpha-helices in the denatured state is higher. Spectrin conformation was found to be very sensitive to the presence of water-soluble organic solvents; the denaturation curves exhibit maxima and minima not typical of protein isothermic denaturation. The observed low conformational stability of spectrin is discussed in the context of its molecular environment and function in the red cell membrane.     

Conformational stability of the polypeptide components of human high density serum lipoprotein.

An investigation of the denaturation of the major polypeptides from human serum high density lipoprotein by guanidine hydrochloride reveals that the lipid-free, water-soluble states are minimally stable relative to the random coil states. These findings are in direct contrast to the resistance of intrinsic membrane proteins (in the absence of ligand) to complete unfolding by the same denaturant. Consideration of the denaturation data for the apolipoproteins together with previously published data from this laboratory on ligand-induced conformational changes indicate that these polypeptides possess several similar conformational states which are readily interconvertible.     

Histone deacetylase complexes: functional entities or molecular reservoirs

Using the dextran-binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD-tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross-linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water-soluble dextran. The purity of the eluted proteins after this one-step affinity purification was 90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins.     

Development and characterization of a novel anti-ceramide antibody

Ceramide is emerging as a key sphingolipid that regulates a variety of cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide. Our results indicate that the antibody (rabbit IgG) specifically recognizes ceramide in lipid overlay assays and detects ceramide species with different fatty acid chain lengths that include C2, C8, C16, C18, C20, and C24. The new antibody was compared with the commercially available anti-ceramide antibody (mouse IgM) in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain the same regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in compartments that are not well identified with the commercially available antibody. In addition to staining of ceramide in protrusions of the plasma membrane, the rabbit IgG also detects ceramide in the Golgi apparatus. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine the localization of ceramide and its interaction with proteins by immunocytochemistry.     

Molecular basis of the heat denaturation of photosystem II

The thermal denaturation of the photosystem II (PSII) membrane protein complex is investigated by assigning the endothermic transitions observed by differential scanning calorimetry (DSC) to the denaturation of particular proteins of the PSII complex. In a prior DSC study of PSII membranes [Thompson, L. K., Sturtevant, J. M., & Brudvig, G. W. (1986) Biochemistry 25, 6161], five DSC peaks were observed in the 30-70 degrees C temperature range (A1, A2, B, C, and D). The A2 peak was assigned to denaturation of a component essential for water oxidation and the B peak to denaturation of a component critical to the remainder of the electron-transport chain. We have now extended these studies with thermal gel analysis and electron paramagnetic resonance (EPR) measurements. Thermal gel analysis, a technique which relies on a change in the solubility properties of a membrane protein upon denaturation, has been used to determine the temperatures of denaturation of all of the major membrane proteins of the PSII complex. EPR experiments have been used to monitor chlorophyll photooxidation and the stability of TyrD+. Peaks B, C, and D in the DSC denaturation profile are each assigned to the denaturation of several proteins, which provides information on the organization of the PSII complex into structural and functional units. Peak B corresponds to the denaturation of peripheral core proteins and closely associated antenna proteins, peak C to the PSII core, and peak D to the loosely associated antenna proteins. No membrane protein is observed to denature during the A2 peak. The A2 peak is altered by the presence of catalase, superoxide dismutase, low chloride, and high pH. These results suggest that the abnormally sharp A2 peak occurs when the highly oxidizing, sequestered Mn complex (the active site in water oxidation) becomes accessible to the aqueous phase, at elevated temperatures. We propose a mechanism for the reaction of the Mn complex with hydroxide ions, which involves peroxide or superoxide and results in the reduction and release of Mn. The proposed model provides insight into the well-known instability of the Mn complex and the role of chloride in stabilizing the complex. This may enable the future development of purification procedures and may explain the sensitivity of the water-oxidizing apparatus of PSII to heat denaturation.     

Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin

Hemifluorinated compounds, such as HF-TAC, make up a novel class of nondetergent surfactants designed to keep membrane proteins soluble under nondissociating conditions [Breyton, C., et al. (2004) FEBS Lett. 564, 312]. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants can coexist with intact lipid vesicles. To test the ability of HF-TAC to assist proper membrane insertion of proteins, we examined its effect on the pH-triggered insertion of the diphtheria toxin T-domain. The function of the T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome. This translocation is accompanied by the formation of a pore, which we used as a measure of activity in a vesicle leakage assay. We have also used F?rster resonance energy transfer to follow the effect of HF-TAC on aggregation of aqueous and membrane-bound T-domain. Our data indicate that the pore-forming activity of the T-domain is affected by the dynamic interplay of two principal processes: productive pH-triggered membrane insertion and nonproductive aggregation of the aqueous T-domain at low pH. The presence of HF-TAC in the buffer is demonstrated to suppress aggregation in solution and ensure correct insertion and folding of the T-domain into the lipid vesicles, without solubilizing the latter. Thus, hemifluorinated surfactants stabilize the low-pH conformation of the T-domain as a water-soluble monomer while acting as low-molecular weight chaperones for its insertion into preformed lipid bilayers.     

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field‐induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Bioelectromagnetics 34:253–263, 2013. © 2012 Wiley Periodicals, Inc.     

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.