Conformational polymorphism, stability and aggregation in spider dragline silks proteins

Spider silk is spun in a complex and unique process, thought to depend on a hydrophobic conversion of a predominantly disordered to a beta-sheet rich protein structures. To test this hypothesis we monitored the effect of cationic (DOTAC) and anionic (alkyl sulfate) detergents and of (ii) solvent polarity using a series of alcohols on the secondary structure transition in dilute solutions of native spidroin. Our results showed that the detergents hydrophilic head charge and hydrophobic tail length cooperatively induced either a transition to the beta-sheet rich form or a stable helical state. Changing the solvent polarity showed that HFIP and TFE induced formation of stable helical forms whereas MeOH, EtOH and IsoP induced a kinetically driven formation of beta-sheet rich structure.     

Amidosulfobetaines, a family of detergents with improved solubilization properties: application for isoelectric focusing under denaturing conditions

The synthesis of a family of new detergents and their use for isoelectric focusing are described. These detergents differ from the conventional sulfobetaines by the presence of an amido group bridging the hydrophobic linear tail and the polar head, which increases their water solubility and urea tolerance considerably. Four different linear alkyl hydrophobic tails were tested, together with three different polar heads. The solubilization of red blood cell ghosts by the 12 resulting detergent performances was evaluated both in solution and in isoelectric focusing runs. Six chemicals gave performances equal or better than those of conventional detergents and could replace them for general use in focusing experiments. In addition, the synthesis of a whole range of compounds allowed valuable insights in the empirical understanding of protein-detergent interactions, which are extensively discussed.     

Enzymology of ubiquinone-utilizing electron transfer complexes in nonionic detergent.

The enzymology of isolated succinate: ubiquinone reductase and ubiquinone: cytochrome c reductase in nonionic detergents (alkyl polyoxyethylene derivatives) was studied. In the membrane the two multiprotein complexes and their hydrophobic substrates ubiquinone and dihydroubiquinone, are embedded in a common lipid bilayer. In detergent solutions the complexes are each inserted into micelles. Detergent micelles also serve as a solvent for the complexes hydrophobic substrates. As a consequence the isolated complexes are in a discontinuous phase with respect to their hydrophobic substrates and with respect to each other. Three types of assays were used. Firstly, single enzyme assays in which the hydrophobic substrates had to transfer from free micelles to the complex-bound micelles in order for enzyme reactions to occur. Secondly, assays in which the enzymic reactions were coupled to auxiliary nonenzymic reactions which rapidly converted the hydrophobic products back into substrates within the complex-bound micelle. Dichloroindophenol was used for the oxidation of dihydroubiquinone and dihydroduroquinone for the reduction of ubiquinone. Thirdly, assays in which the succinate: ubiquinone reductase reaction was coupled with the ubiquinone: cytochrome c reductase reaction. With the first type of assay, the kinetics of the substrate transfer reaction was dependent upon the type of detergent. In detergents with small polyoxyethylene head groups the transfer reactions were rate-limiting, and in detergents with large polyoxyethylene head groups the transfer reactions were fast and the enzymic reactions were rate-limiting...     

An evaluation of the hydrophobic interactions of chick muscle acetylcholinesterase by charge shift electrophoresis and gradient centrifugation

The hydrophobic interactions of globular forms of acetylcholinesterase from adult and embryonic chick muscles have been analyzed by sucrose gradient centrifugation and non denaturing polyacrylamide gel electrophoresis. The presence of positively- or negatively-charged detergents influences the electrophoretic migrations of hydrophobic globular forms, whereas the mobility of hydrophilic components is unchanged. We defined an hydrophobicity index (HI) which quantitatively reflects this interaction.Globular forms of acetylcholinesterase were isolated in preparative sucrose gradients of muscle extracts. The G₁ form (5 S) appeared as a single band in electrophoresis, the G₂ form (7 S) under two and the G₄ form (11 S) under three electromorphs. The G₁ and the G₂ forms interacted with detergents: this resulted in a shift in their sedimentation in sucrose gradients upon removal of detergents, and in a modification of their electrophoretic migrations in the presence of charged detergents (HI = 1.0 for G₁, HI = 1.7 for G₂). The G₄ form was heterogenous: one band (G_4f) did not interact with detergent (HI = 0.1). The other variants (G_4i and G_4s) were clearly hydrophobic (HI = 0.5 and HI = I respectively). The hydrophilic and hydrophobic variants of the G₄ form however, were not resolved by sedimentation analysis performed in the presence of Triton X100, but their separation was improved in the presence of 10-oleyl-ether. Therefore, the combination of electrophoretic and sedimentation methods, as described in this paper, can be used successfully for subdividing a single molecular form (size isomer defined by hydrodynamic parameters) into several constituents differing by their hydrophobic interactions.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI greater than 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112-120) gives good resolution of proteins of pI 5-8, including cytochromes P-450, but fails to resolve a major acidic protein of pI less than 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R . N+(CH3)2 . CH2CH2CH2SO3- are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C14 or C16) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Mechanism of Gemini Disulfide Detergent Mediated Oxidative Refolding of Lysozyme in a New Artificial Chaperone System

Gemini surfactants are a new class of surfactants that consist of two hydrophilic head groups and two hydrophobic tails separated by a spacer group. As the properties of geminis are different to their monomeric counterparts, a large number of applications have been investigated. Here we report on the use of a new class of gemini detergents containing a disulfide bond in the spacer (Det-SS-Det) for protein refolding. Using lysozyme as a model protein we could demonstrate that the disulfide gemini detergents allow oxidative refolding of the protein in the absence of any external redox system in an “artificial chaperone system”. Refolding kinetics using gemini disulfide detergents differing in their hydrophobicity were analysed to determine the folding and aggregation rate constants. The results point to an important role of the transiently formed mixed disulfides between the protein and the detergent (Prot-SS-Det) in the oxidative refolding process of lysozyme.     

Extraction of detergents from hydrophobic proteins with isopentanol: application to electrophoretic analysis of photosynthetic bacterial hydrophobic proteins

The method for extracting Triton X-100 used by I. H. Mather and C. B. Tampling [Anal. Biochem. 93, 139-142 (1979)], has been extended to other detergents of different charge and chemical nature. All the detergents tested can be extracted with isopentanol in conditions in which not more than 8% of hydrophobic or hydrophilic protein is lost from the water phase. The removal of detergent from reaction centers and light harvesting protein-pigment complexes of photosynthetic bacteria, eliminates the artifacts of oligomers when analyzed by sodium dodecyl sulfate-gel electrophoresis.     

Influence of detergent polar and apolar structure upon the temperature dependence of beef heart cytochrome c oxidase activity

The temperature dependence of lipid-depleted beef heart cytochrome c oxidase activity was studied in a series of chemically homogeneous detergents. The detergents that were tested included C10 to C18 maltosides, C8 to C12 glucosides, C8 to C16 Zwittergents, and C12 poly(oxyethylene) ethers. The observed rates of electron transport were dependent upon the structure of the polar head group and the length of the hydrocarbon tail. Of the detergents tested, the alkyl maltosides were the best in terms of both high rates of electron transport and superior enzyme stability. With the maltosides, changing the length of the alkyl tail affected the activity of cytochrome c oxidase in a manner quite similar to that reported with synthetic phosphatidylcholines and phosphatidylethanolamines [Vik, S. B., & Capaldi, R. A. (1977) Biochemistry 16, 5755-5759], suggesting that the alkyl maltosides can mimic some of the features of the membrane environment. In each of the detergents, the activation enthalpy (determined from the slope of an Arrhenius plot) was nearly identical, suggesting that the same electron-transfer step within cytochrome c oxidase is rate limiting. This result has been interpreted as evidence for the existence of two or more conformers of cytochrome c oxidase, one of which is significantly more active than the other(s). The enzyme turnover number, which changes by 2 orders of magnitude depending upon the structure of the bound detergent, may reflect the ability of each detergent to alter the equilibrium between the active and nearly inactive conformers.     

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.     

Oblique self-assemblies and order-order transitions in polypeptide complexes with PEGylated triple-tail lipids

We report on highly ordered oblique self-assemblies in ionic complexes of PEGylated triple-tail lipids and cationic polypeptides, as directed by side-chain crystallization, demonstrating also reversible oblique-to-hexagonal order-order transitions upon melting of the side chains. This is achieved in bulk by complexing cationic homopolypeptides, poly-l-lysine (PLys), poly-l-arginine (PArg), and poly-l-histidine (PHis), in stoichiometric amounts with anionic lipids incorporating two hydrophobic alkyl tails and one hydrophilic polyethylene glycol (PEG) tail in a star-shaped A(2)B geometry. Based on Fourier transform infrared spectroscopy (FTIR), the PLys and PArg complexes fold into α-helical conformation. Aiming to periodicities at different length scales, that is, hierarchies, the PEG tails were selected to control the separation of the polypeptide helices in one direction while the alkyl tails determine the distance between the hydrophilic polypeptide/PEG layers, resulting in an oblique arrangement of the helices. We expect that the high overall order, where the self-assembled domains are in 2D registry, is an outcome of a favorable interplay of plasticization due to the hydrophobic and hydrophilic lipid tails combined with the shape persistency of the peptide helices and the crystallization of the lipid alkyl chains. Upon heating the complexes over the melting temperature of the alkyl tails, an order-order transition from oblique to hexagonal columnar morphology was observed. This transition is reversible, that is, the oblique structure with 2D correlation of the helices is fully returned upon cooling, implying that the alkyl tail crystallization guides the structure formation. Also PHis complex forms an oblique self-assembly. However, instead of α-helices, FTIR suggests formation of helical structures lacking intramolecular hydrogen bonds, stabilized by steric crowding of the lipid. The current study exploits competition between the soft and harder domains, which teaches on concepts toward well-defined polypeptide-based materials.     

Controlled template-assisted assembly of plasmid DNA into nanometric particles with high DNA concentration

A series of novel cationic detergents that contain cleavable hydrophilic isothiuronium headgroups was synthesized, and their utility in controlled assembly of plasmid DNA into small stable particles with high DNA concentration investigated. The detergents have alkyl chains of C(8)-C(12) and contain hydrophilic isothiuronium headgroups that give relatively high critical micelle concentration (CMC) to the detergents (>10 mM). The isothiuronium group masks a sulfhydryl group on the detergent and can be cleaved in a controlled manner under basic conditions to generate a reactive thiol group. The thiol group can undergo a further reaction after the detergents have accumulated on a DNA template to form a disulfide-linked lipid containing two alkyl chains. The pH-dependent kinetics of cleavage of the isothiuronium group, the CMC of the surfactants, the formation of the complexes, and the transfection efficiency of the DNA complexes have been investigated. Using the C(12) detergent, a approximately 6 kilo-basepair plasmid DNA was compacted into a small particle with an average diameter of around 40 nm with a approximately -13 mV zeta-potential at high DNA concentration (up to 0.3 mg/mL). The compounds were well tolerated in cell culture and showed no cytotoxicity under their CMCs. Under appropriate conditions, the small particle retained transfection activity.     

去垢剂在膜蛋白研究中的应用

去垢剂是同时具有亲水极性基团和疏水非极性基团的双极性分子,能够使脂膜解体释放膜蛋白,并在溶液中为去膜状态下的膜蛋白提供疏水环境,维持和保护膜蛋白的疏水跨膜结构,在膜蛋白的结构和功能研究中有重要的意义。去垢剂的双极性和理化特性,如临界胶束浓度能够极大影响去垢剂和膜蛋白间的相互作用。在膜蛋白研究中,需要充分利用去垢剂的结构和特性:一方面,需要利用去垢剂代替脂质分子支持和稳定去膜状态下膜蛋白的结构和功能;另一方面,需要控制去垢剂和膜蛋白的相互作用,以满足膜蛋白结构研究如蛋白质结晶试验的要求。简要介绍了去垢剂在膜蛋白研究中的最新应用进展,涉及去垢剂在膜蛋白离体表达、分离和纯化、以及结构研究中的应用。     

Special considerations in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1): solubilization of SAT-1 with lauryldimethylamine oxide

Lauryldimethylamine oxide (LDAO) was employed in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1) (4). This detergent has advantages over the typically employed Triton detergents in the solubilization and stabilization of this sialyltransferase. Crude protein fractions solubilized from rat liver Golgi by several such detergents are very similar in composition as determined by two-dimensional gel electrophoresis. However, LDAO appears to activate and stabilize SAT-1 activity. It is possible that SAT-1 activation involves the structurally similar hydrophobic moieties and quaternary amino groups of LDAO and phosphatidylcholine.     

Evaluation of lipoxygenase inhibitory activity of anacardic acids

6-Alkylsalicylic acids inhibit the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) competitively and without pro-oxidant effects. This activity is largely dependent on the nature of their alkyl side chains. Inhibitory activities of anacardic acids, viz. 6-pentadec(en)ylsalicylic acids, isolated from the cashew Anacardium occidentale, were initially used for comparison because their aromatic head portions are the same. Consequently, the data should be interpreted to mean that changes in the hydrophobic side chain tail portions of the molecules evaluated correlate with the specific activity determined.     

Disturb or Stabilize? A Molecular Dynamics Study of the Effects of Resorcinolic Lipids on Phospholipid Bilayers

Resorcinolic lipids, or resorcinols, are commonly found in plant membranes. They consist of a substituted benzene ring forming the hydrophilic lipid head, attached to an alkyl chain forming the hydrophobic tail. Experimental results show alternative effects of resorcinols on lipid membranes. Depending on whether they are added to lipid solutions before or after the formation of the liposomes, they either stabilize or destabilize these liposomes. Here we use atomistic molecular dynamics simulations to elucidate the molecular nature of this dual effect. Systems composed of either one of three resorcinol homologs, differing in the alkyl tail length, interacting with dimyristoylphosphatidylcholine lipid bilayers were studied. It is shown that resorcinols preincorporated into bilayers induce order within the lipid acyl chains, decrease the hydration of the lipid headgroups, and make the bilayers less permeable to water. In contrast, simulations in which the resorcinols are incorporated from the aqueous solution into a preformed phospholipid bilayer induce local disruption, leading to either transient pore formation or even complete rupture of the membrane. In line with the experimental data, our simulations thus demonstrate that resorcinols can either disturb or stabilize the membrane structure, and offer a detailed view of the underlying molecular mechanism.     

Amphiphilic properties of the avian myeloblastosis virus major glycoprotein, gp 80

The major glycoprotein (gp 80) from avian myeloblastosis virus (AMV) displays significant lipophilic properties, as shown by its strong interactions with acetylated uncharged decylamino agarose in hydrophobic chromatography. In effect, release from binding was achieved only by the added presence of a polarity reducing agent (ethylene glycol) and the strong anionic detergent sodium dodecyl sulfate. The hydrophobic behavior of the glycoprotein, coupled to the high content of hydrophilic carbohydrates, indicates its amphiphilic character. Confirmation of the amphiphilic nature of the AMV gp 80 was obtained by charge shift electrophoresis and crossed hydrophobic interaction immunoelectrophoresis. In both instances, the electrophoretic behavior of the glycoprotein was dependent on the presence of detergents. The AMV gp 80 displays the properties of integral membrane proteins.     

Two-dimensional electrophoresis of membrane proteins factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI > 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112–120) gives good resolution of proetins of pI 5–8, including cytochromes P-450, but fails to resolve a major acidic protein of pI < 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R·N⁺(CH₃)₂·CH₂CH₂CH₂SO₃^? are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C₁₄ or C₁₆) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.     

Biosynthesis and structure of membrane and secretory immunoglobulins

Summary Almost all of the body's extracellular immunoglobulin (Ig) is derived from Ig-secreting plasma cells of lymphoid tissues. The secreted material is a heterogeneous mixture of different classes and specificities. Lymphoid tissues also contain a large number of essentially non-secretory cells — B lymphocytes — which bear Ig firmly associated with their plasma membranes. Ig molecules thus exist in two functionally different forms, as membrane-bound antigen receptors on the surface of B lymphocytes on the one hand, and as humoral secreted Ig antibodies on the other. On B cells, membrane-bound heavy chains have an apparent mol. wt. slightly larger than that of secreted heavy chains from plasma cells. Membrane-bound but not secreted heavy chains bind detergents, thus suggesting the presence of a hydrophobic region in membrane-bound heavy chains, which is absent in secreted heavy chains. Most investigations have dealt with immunoglobulin M. The two types of IgM heavy chains differ at their carboxy termini. Recent investigations at the nucleic acid level demonstrate that membrane-associated µ chains contain a 41-residue hydrophobic tail adjacent to the last constant domain, whereas secretory µ chains contain a 20-residue hydrophilic tail. At the present time, evidence is accumulating that all membrane-bound Ig heavy chain classes may contain similar hydrophobic structures necessary for anchorage of the molecules into the lipid bilayer.