NMR studies of the influence of dodecyl sulfate on the amide hydrogen exchange kinetics of a micelle-solubilized hydrophobic tripeptide

Backbone amide hydrogen exchange measurements are an important source of information about the internal dynamics of proteins. Before such measurements can be interpreted unambiguously, contributions to hydrogen exchange rates from the chemical and physical environment of the amides must be taken into account. Membrane proteins are often solubilized in detergents, yet there have not been any systematic investigations of the possible effects detergents may have on the amide hydrogen exchange rates of proteins. To address this question, we have measured individual backbone and carboxyl-terminal amide exchange rates for the amphipathic tripeptide Leu-Val-Ile-amide dissolved in water and dodecyl sulfate micelles. 1H NMR spectroscopy was used to measure exchange using the direct exchange-out into D2O technique at 5 degrees C and using an indirect steady-state saturation-transfer technique at 25 degrees C. The broadening effect of micelle-incorporated spin-labeled fatty acid (12-doxylstearate) on the 1H NMR spectra of both the detergent and the peptide resonances was used to demonstrate that the tripeptide is intimately associated with the micelle. The resonance from formate ion, which is excluded from the micelle, was unperturbed by the spin label. The detergent did not retard the exchange rates of either the primary (terminal) or secondary (backbone) amides of the tripeptide. This suggests that the micelle/peptide interaction does not restrict access of charged catalysts and water to these amides and shows that the peptide amides are not hydrogen bonded. However, the pH for the exchange minima of these amides in detergent was increased between 1.2 and 1.7 units compared to exchange in water.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

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.     

Activated-Sludge Nitrification in the Presence of Linear and Branched-Chain Alkyl Benzene Sulfonates

The effects of biodegradable linear alkyl benzene sulfonate and branched-chain alkyl benzene sulfonate detergents on activated-sludge nitrification were investigated by administering a synthetic waste containing up to 23 mg of each detergent per liter to eight bench-scale, batch, activated-sludge units. It was found that both detergents tended to promote complete oxidation of ammonia to nitrate, whereas control units produced approximately equal amounts of nitrite and nitrate. Various hypotheses are offered to explain the phenomenon.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

Protein conformational changes and myelin solubilization by anion-detergent solutions.

The addition of sodium sulfate to a myelin suspension in sodium phosphate buffer at neutral pH, containing octyl glucoside detergent (OG), increases the membrane solubility more than 5-fold by an unknown structural mechanism. FTIR spectroscopy has been applied to investigate anion effects on the conformational structure of myelin proteins. Sulfate and sulfate-phosphate media, but not phosphate alone, induce a great conformational protein disorder. The addition of the detergent to the anion mixture solution prevents the myelin from protein denaturation. The conformational transitions have also been quantified through the amide I region. Explanations of these changes and their connections with myelin solubility are also included.     

Alcohol-induced denaturation of beta-lactoglobulin: a close correlation to the alcohol-induced alpha-helix formation of melittin

Alcohols denature the native structure of proteins and induce alpha-helical structure. The potential of alcohols causing such effects varies substantially depending on the alcohol species. With beta-lactoglobulin as a model protein, we compared the effects of various alcohols and observed the additive contribution of each group constituting the alcohol molecules. Whereas the hydrophobic hydrocarbon group promotes the transition according to their size, hydrophilic hydroxyl group suppresses the transition. Halogen groups promote the transition depending on their type and number. It has been known that alcohols induce the alpha-helical structure on the short peptides such as melittin. There is a close correlation between the potentials of alcohol in denaturing beta-lactoglobulin and those in inducing the helical structure in melittin, indicating that the underlying mechanisms of the two phenomena are the same.     

High water solubility and fold in amphipols of proteins with large hydrophobic regions: oleosins and caleosin from seed lipid bodies

Seed lipid bodies constitute natural emulsions stabilized by specialized integral membrane proteins, among which the most abundant are oleosins, followed by the calcium binding caleosin. These proteins exhibit a triblock structure, with a highly hydrophobic central region comprising up to 71 residues. Little is known on their three-dimensional structure. Here we report the solubilization of caleosin and of two oleosins in aqueous solution, using various detergents or original amphiphilic polymers, amphipols. All three proteins, insoluble in water buffers, were maintained soluble either by anionic detergents or amphipols. Neutral detergents were ineffective. In complex with amphipols the oleosins and caleosin contain more beta and less alpha secondary structures than in the SDS detergent, as evaluated by synchrotron radiation circular dichroism. These are the first reported structural results on lipid bodies proteins maintained in solution with amphipols, a promising alternative to notoriously denaturing detergents.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Dodecyl Maltoside Protects Membrane Proteins In Vacuo

Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a β-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The β-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an α-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.     

Effects of chaotropic agents versus detergents on dihydroorotate dehydrogenase.

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

Inhibition of collagenase from Clostridium histolyticum by phosphoric and phosphonic amides

Di- and tripeptides with sequences present in collagen that are known to occupy the S1' through S3' subsites at the active site of the collagenase from Clostridium histolyticum do not themselves inhibit this zinc protease. Thus glycylproline, glycylprolylalanine, and their C-terminal amides are not inhibitors. N alpha-Phosphorylglycylproline, N alpha-phosphorylglycyl-L-prolyl-L-alanine, and their C-terminal amides are weak inhibitors with IC50's (concentration causing half-maximal inhibition) of 4.6, 0.8, 3, and 1.5 mM, respectively. Extension of glycyl-L-prolyl-L-alanine to L-leucyl-glycyl-L-prolyl-L-alanine gives a tetrapeptide known to occupy the S1, S1', S2', and S3' subsites of collagenase when present in collagen but that still does not itself inhibit the enzyme. (Isoamylphosphonyl)glycyl-L-prolyl-L-alanine, a peptide containing a tetrahedral phosphorus atom at the position of the amide carbonyl carbon of the L-leucylglycyl amide bond of the parent tetrapeptide, inhibits collagenase with an IC50 of 16 microM, at least 1000-fold more potent than the parent peptide. Substitution of the two-carbon ethyl chain of alanine for the five-carbon isoamyl chain of leucine increases the IC50 to 46 microM. Substitution of the n-decyl chain for the isoamyl chain does not change the IC50. (Isoamylphosphonyl)glycyl-glycyl-L-proline contains a tripeptide that does not occupy the S1' through S3' subsites of collagenase when this peptide is present in collagen and thus has an IC50 of 4.4 mM. (Isoamylphosphonyl)glycyl-L-prolyl-L-alanine may be an analogue of the tetrahedral transition state for the hydrolysis of the natural collagen substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation of the Detergent-Binding Capacity and Phospholipid Content of Membrane Proteins When Purified in Different Detergents

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.     

Dodecyl Maltoside Protects Membrane Proteins In Vacuo

Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a β-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The β-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an α-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.     

Studies involving the electrostatic potential of valinomycin

The electrostatic potential of valinomycin in various conformations as obtained by the crystal structures (uncomplexed, complexed) and theoretical considerations have been evaluated and compared. The potential energy profiles along the æ axis of the bracelet-like structures show a systematic variation from the uncomplexed to the complexed structure. This type of conformational change and the potential variation are probably associated with different states of ion transport, like the capture and release of ions by the ionophore. Also, the asymmetry of the molecule due to D-HyIV on one side and L-Lac on the other side is reflected in the potential values along the Z-axis, the magnitude of which, is considerable in the uncomplexed structure. The evaluation of the potential at the ab-initio level on smaller fragments indicate that the order of liganding capacity of oxygen is amide > ether > ester. Also, the inductive effects due to alkyl substitution is negligible as evidenced by the potential studies on the substituted amides and esters.     

Tertiary structural constraints on protein evolutionary diversity: templates, key residues and structure prediction

The pattern of residue substitution in divergently evolving families of globular proteins is highly variable. At each position in a fold there are constraints on the identities of amino acids from both the three-dimensional structure and the function of the protein. To characterize and quantify the structural constraints, we have made a comparative analysis of families of homologous globular proteins. Residues are classified according to amino acid type, secondary structure, accessibility of the sidechain, and existence of hydrogen bonds from sidechain to other sidechains or peptide carbonyl or amide functions. There are distinct patterns of substitution especially where residues are both solvent inaccessible and hydrogen bonded through their sidechains. The patterns of residue substitution can be used to construct templates or to identify 'key' residues if one or more structures are known. Conversely, analysis of conversation and substitution across a large family of aligned sequences in terms of substitution profiles can allow prediction of tertiary environment or indicate a functional role. Similar analyses can be used to test the validity of putative structures if several homologous sequences are available.     

Interaction of the mouse and bovine myelin basic proteins and two cleavage fragments with anionic detergents

The binding of deoxycholate and dodecyl sulfate to the mouse and bovine myelin basic proteins and two peptide fragments, obtained by cleavage of the bovine basic protein at its single tryptophan residue, was examined. Complete equilibrium binding isotherms for both detergents were obtained by examining their binding to each of the polypeptides immobilized on agarose. The bulk of the binding of dodecyl sulfate was found to be highly cooperative, and at saturation all four polypeptides bound far more detergent than globular, water-soluble proteins. The sum of the dodecyl sulfate bound by each of the two bovine basic protein cleavage fragments was almost twice that bound by the intact protein at saturation, suggesting that cleavage of the bovine basic protein exposes sites for additional binding of dodecyl sulfate. At pH values below pH 8.0, an additional cooperative transition was observed below the critical micelle concentration of sodium dodecyl sulfate in the binding isotherms of all four polypeptides. The midpoint of this transition corresponded to an apparent pK of approximately 5.5; however, the destruction of 90% of the histidine residues in the bovine basic protein had no effect on this transition. At pH 9.2 and moderate ionic strength (I = 0.1), the bulk of the binding of deoxycholate to the mouse and bovine basic proteins occurred at and above the critical micelle concentration of the detergent; and saturation values of deoxycholate binding to these two proteins were considerably higher than that reported for globular, water-soluble proteins. In marked contrast to the results with dodecyl sulfate, neither cleavage fragment was observed to bind deoxycholate. The results suggest that the higher ordered structure of the bovine basic protein may play an important role in the binding of anionic amphiphiles to the protein.     

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.     

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.