Labelling of the intramembranous region of the major sialoglycoprotein of human erythrocytes with a photosensitive hydrophobic probe.

Human erythrocyte membranes were incubated with the photosensitive hydrophobic reagent 1-azido-r-iodo[3H]benzene and the mixture was irradiated. The major sialoglycoprotein was then isolated and the labelled polypeptide subjected to proteolytic dissection. Characterization of the purified tryptic and chymotryptic peptides show that the probe is covalently attached only to the transmembrane region of the protein. This labelling pattern is discussed in relation to the use of such reagents for the identification of segments of membrane proteins exposed to the hydrophobic millieu of the membrane.     

Computational differentiation of N-terminal signal peptides and transmembrane helices

Signal peptides and transmembrane helices both contain a stretch of hydrophobic amino acids. This common feature makes it difficult for signal peptide and transmembrane helix predictors to correctly assign identity to stretches of hydrophobic residues near the N-terminal methionine of a protein sequence. The inability to reliably distinguish between N-terminal transmembrane helix and signal peptide is an error with serious consequences for the prediction of protein secretory status or transmembrane topology. In this study, we report a new method for differentiating protein N-terminal signal peptides and transmembrane helices. Based on the sequence features extracted from hydrophobic regions (amino acid frequency, hydrophobicity, and the start position), we set up discriminant functions and examined them on non-redundant datasets with jackknife tests. This method can incorporate other signal peptide prediction methods and achieve higher prediction accuracy. For Gram-negative bacterial proteins, 95.7% of N-terminal signal peptides and transmembrane helices can be correctly predicted (coefficient 0.90). Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 99% (coefficient 0.92). For eukaryotic proteins, 94.2% of N-terminal signal peptides and transmembrane helices can be correctly predicted with coefficient 0.83. Given a sensitivity of 90%, transmembrane helices can be identified from signal peptides with a precision of 87% (coefficient 0.85). The method can be used to complement current transmembrane protein prediction and signal peptide prediction methods to improve their prediction accuracies.     

Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry

The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins.     

Large scale extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey by precipitation with polyethylene glycol and partitioning in aqueous two-phase systems.

The milk proteins alpha-lactalbumin and beta-lactoglobulin have been isolated from bovine whey by fractional precipitation with polyethylene glycol (PEG) and hydrophobic partitioning in aqueous PEG-hydroxypropylstarch two-phase systems using PEG-bound palmitate as hydrophobic ligand. The possible use of this combination for large scale purification of these whey proteins is discussed.     

Studies of the minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes

The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.     

Separation of Listeria monocytogenes and Salmonella berta from a complex food matrix by aqueous polymer two-phase partitioning

In the present study, the use of aqueous polymer two-phase systems for separation of pathogenic bacteria from a complex food sample was investigated. Three different two-phase systems, a polyethylene glycol 3350/dextran T 500, a methoxy polyethylene glycol 5000/dextran T 500 and a polyethylene glycol 3350/hydroxypropyl starch system, were compared at pH 3 and pH 6 for their capacity to separate the pathogenic bacteria Listeria monocytogenes and Salmonella berta from a Cumberland sausage. In all three phase systems, the food particles partitioned to the lower phase. Best performance was obtained by the polymer combinations, polyethylene glycol 3350/dextran T 500 and polyethylene glycol 3350/hydroxypropyl starch. In these systems, Salmonella berta partitioned to the hydrophobic upper phase both at pH 3 and pH 6 with an average partitioning ratio of 80% and a recovery of 56%. Listeria monocytogenes partitioned to the upper phase at pH 3 only with an average partitioning ratio of 72% and a recovery of 45%. This method may become a valuable tool for separation of bacteria from complex food matrices.     

Affinity‐Triggered Assemblies Based on a Designed Peptide–Peptide Affinity Pair

Affinity‐triggered assemblies rely on affinity interactions as the driving force to assemble physically crosslinked networks. WW domains are small hydrophobic proteins binding to proline‐rich peptides that are typically produced in the insoluble form. Previous works attempted the biological production of the full WW domain in tandem to generate multivalent components for affinity‐triggered hydrogels. In this work, an alternative approach is followed by engineering a 13‐mer minimal version of the WW domain that retains the ability to bind to target proline‐rich peptides. Both ligand and target peptides are produced chemically and conjugated to multivalent polyethylene glycol, yielding two components. Upon mixing together, they form soft biocompatible affinity‐triggered assemblies, stable in stem cell culture media, and display mechanical properties in the same order of magnitude as for those hydrogels formed with the full WW protein in tandem.     

Optimizing synthesis and expression of transmembrane peptides and proteins

Structural studies of full-length membrane proteins have been hindered by their hydrophobicity and low expression in a variety of systems. However, a simplifying aspect of membrane protein folding is that individual transmembrane segments or membrane protein fragments have been observed to represent independent folding domains, and as such, can facilitate the study of packing interactions between TM helices, and the collection of structural information regarding membrane proteins. This review focuses on two categories of techniques--total peptide synthesis and bacterial expression--that can each be optimized for preparation of transmembrane protein segments. First, synthesis of hydrophobic transmembrane peptides that are N- and/or C-tagged with solubilizing residues such as lysine can improve manipulation of the transmembrane core in a variety of biophysical experiments. In this context, we describe general protocol considerations during the synthesis, cleavage, and purification stages of these peptides to identify appropriate parameters that combine to improve yields of hydrophobic peptides. Second, bacterial expression of membrane protein fragments is a useful tool for producing large quantities of hydrophobic protein segments. Targeting protein expression within Escherichia coli can facilitate purification, while attaching the hydrophobic construct to a hydrophilic fusion protein can amplify expression. We show that adapting protein constructs to comply with expression host specifications, in concert with thorough exploration of expression conditions such as the type of media used for expression, temperature, and cell strain, can significantly improve protein yields.     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

The effect of a range of biological polymers and synthetic surfactants on the adhesion of a marine Pseudomonas sp. strain NCMB 2021 to hydrophilic and hydrophobic surfaces

Abstract The effect of a range of biological polymers and synthetic surfactants on the adhesion of a marine Pseudomonas sp. strain NCMB2021 to hydrophilic glass and hydrophobic polystyrene has been investigated. Brij 56 (polyethylene oxide (10) cetyl ether) was the only compound that had a significant effect, almost totally inhibiting the adhesion of Pseudomonas sp. NCMB2021 to hydrophobic polystyrene, but having little or no effect on hydrophilic glass. The surfactant was demonstrated to be effective both when present in the bacterial suspension at low concentrations (approx. 5 ppm), and when pre-adsorbed onto the substratum. Brij 56 was shown to prevent the adhesion of a range of marine and fresh-water bacteria to polystyrene.
It is proposed that on a hydrophobic substratum Brij 56 is adsorbed via its hydrophobe in such a way that the polyethylene glycol chains are pointing outwards into the aqueous phase giving a surface with a high density of uncharged, highly hydrated hydrophilic chains, forming a steric barrier which inhibits the adhesion of bacteria.     

Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes.

EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cys-less) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway.     

Peptide-polyethylene glycol conjugates: synthesis and properties of peptides bearing a C-terminal polyethylene glycol chain

The introduction of a polyethylene glycol chain has become a popular tool for increasing water solubility and bioavailability. Our interest in the development of catalytically active peptides and the selective recognition of peptides has led us to investigate strategies to increase the solubility of peptides in organic solvents. Specifically, we became interested in the introduction of solubilizing moieties at the C-terminus of two peptides. Here we present different synthetic strategies for the preparation of peptide-polyethylene glycol conjugates and discuss the effect of the polyethylene glycol chain on the solubility and other properties, such as the catalytic activity of these peptides.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Aqueous two-phase partition of complex protein feedstocks derived from brain tissue homogenates

This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).     

Differentiation of lipid-associating helices by use of three-dimensional molecular hydrophobicity potential calculations.

Several types of lipid-associating helices exist: transmembrane helices such as in receptor proteins, pore-forming helices in ion channel proteins, fusion-inducing peptides in viral proteins, and amphipathic helices such as in plasma apolipoproteins. In order to propose a classification of these helices according to their molecular properties, we introduce the concept of molecular hydrophobicity potential for such helical segments. The calculation of this parameter for alpha-helices enables the visualization of the hydrophobic and hydrophilic envelopes around the peptide and their three-dimensional representation by molecular graphics. We have used this parameter to differentiate between pore-forming helices with a hydrophobic envelope larger than the hydrophilic component, membrane-spanning helices surrounded almost entirely by an hydrophobic envelope, fusiogenic peptides with an hydrophobicity gradient both around the helix and along the axis, and finally, amphipathic helices with a predominantly hydrophilic envelope. The structure of the lipid-protein complexes is determined by a number of different interactions: the hydrophobic interaction of the apolar faces of the helices with lipids, the polar interaction of the hydrophilic sides of different helices with each other, and the interaction of hydrophilic residues with the aqueous solvent. The relative magnitude of the hydrophobic and hydrophilic envelopes accounts for the differences in the structure of the lipid-protein complexes. Purely hydrophobic interactions stabilize transmembrane helical segments, while hydrophobic interactions with the lipid phase and with each other are involved in the stabilization of the pore-forming helices. In contrast, both hydrophobic interactions with the lipids and hydrophilic interactions with the aqueous phase contribute to the arrangement of amphipathic helices around the edges of the discoidal lipid-apoprotein complexes.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

Biosynthesis of cholesterol linoleate by polyethylene glycol-modified cholesterol esterase in organic solvents.

Cholesterol esterase modified with polyethylene glycol was able to dissolve in some highly hydrophobic solvents such as benzene and toluene, and catalyze the synthesis of cholesterol linoleate with time dependency in the reverse of the usual reaction in aqueous solvents. Enzymatic cholesterol linoleate synthesis followed Michaelis-Menten kinetics which depended on the concentration of cholesterol and linoleic acid. When more than 20 mM of both substrates was used, the specific activity in this esterification was 200-250 nmol/min/mg protein. The apparent Km value for cholesterol and linoleic acid was 3.7 and 7.6 mM, respectively. The possibility of using such a modified enzyme for the synthesis of less stable cholesterol esters is discussed.     

Structural characterization of Na,K-ATPase from shark rectal glands by extensive trypsinization

Extensive trypsinization of Na,K-ATPase from the salt gland of Squalus acanthias removes about half of the extramembranous protein mass of the alpha-subunit, while leaving the beta-subunit intact. Sequence analysis and epitope recognition of the remaining alpha-peptides show that transmembrane segments M1/M2 and M3/M4 are present when trypsinization is performed in either NaCl or RbCl. The M5/M6 segment and the intact 19-kDa peptide (M7-M10) are detected in Rb-trypsinized membranes but not in Na-trypsinized membranes. The L7/L8 loop is associated with Na-trypsinized membranes, indicating the presence of an M7/M8 or M8/M9 fragment. Freeze-fracture electron microscopy of both Rb- and Na-trypsinized membranes reveals intramembranous particles that indicate a retained cluster of peptides, even in the absence of an intact 19-kDa fragment. The rotational diffusion of covalently spin-labeled trypsinized complexes is studied in the presence of poly(ethylene glycol) or glycerol by using saturation transfer electron spin resonance. Rotational correlation times in aqueous poly(ethylene glycol) are longer than in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that poly(ethylene glycol) induces aggregation of the tryptic peptides (and beta-subunit) within the membrane. The aggregates of enzyme trypsinized in the presence of NaCl are larger than those for enzyme trypsinized in RbCl, at both low and high aqueous viscosities. Similarities in mobility for native and Rb-trypsinized enzymes suggest either a change in average orientation of the spin-label upon trypsinization or that trypsinization leads to a reorganized protein structure that is more prone to aggregation.     

Protein–lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring (Review)

Biological membranes are characterized by a heterogeneous composition, which is not only manifested in the wide variety of their components, but also in aspects like the lateral organization, topology, and conformation of proteins and lipids. In bringing about the correct membrane structure, protein–lipid interactions can be expected to play a prominent role. The extent of hydrophobic matching between transmembrane protein segments and lipids potentially constitutes a versatile director of membrane organization, because a tendency to avoid hydrophobic mismatch could result in compensating adaptations such as tilt of the transmembrane segment or segregation into distinct domains. Also, interfacial interactions between lipid headgroups and the aromatic and charged residues that typically flank transmembrane domains may act as an organizing element. In this review, we discuss the numerous model studies that have systematically explored the influence of hydrophobic matching and interfacial anchoring on membrane structure. Designed peptides consisting of a polyleucine or polyleucine/alanine hydrophobic stretch, which is flanked on both sides by tryptophan or lysine residues, reflect the general layout of transmembrane protein segments. It is shown for phosphatidylcholine bilayers and for other model membranes that these peptides adapt a transmembrane topology without extensive peptide or lipid adaptations under conditions of hydrophobic matching, but that significant rearrangements can result from hydrophobic mismatch. Moreover, these effects depend on the nature of the flanking residues, implying a modulation of the mismatch response by interfacial interactions of the flanking residues. The implications of these model studies for the organization of biomembranes are discussed in the context of recent experiments with more complex systems.