Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study

Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.     

The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media

Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.     

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.     

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.     

Amphipols in G Protein-Coupled Receptor Pharmacology: What Are They Good For?

G protein-coupled receptors are at a central node of all cell communications. Investigating their molecular functioning is therefore crucial for both academic purposes and drug design. However, getting the receptors as isolated, stable and purified proteins for such studies still stumbles over their instability out of the membrane environment. Different membrane-mimicking environments have been developed so far to increase the stability of purified receptors. Among them are amphipols. These polymers not only preserve the native fold of receptors purified from membrane fractions but they also allow specific applications such as folding receptors purified from inclusion bodies back to their native state. Of importance, amphipol-trapped G protein-coupled receptors essentially maintain their pharmacological properties so that they are perfectly adapted to further investigate the molecular mechanisms underlying signaling processes. We review here how amphipols have been used to refold and stabilize detergent-solubilized purified receptors and what are the main subsequent molecular pharmacology analyses that were performed using this strategy.     

Molecular Dynamics Simulations of a Membrane Protein/Amphipol Complex

Amphipathic polymers known as “amphipols” provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.     

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.     

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

Discovering how membrane proteins recognize signals and passage molecules remains challenging. Life depends on compartmentalizing these processes into dynamic lipid bilayers that are technically difficult to work with. Several polymers have proven adept at separating the responsible machines intact for detailed analysis of their structures and interactions. Styrene maleic acid (SMA) co-polymers efficiently solubilize membranes into native nanodiscs and, unlike amphipols and membrane scaffold proteins, require no potentially destabilizing detergents. Here we review progress with the SMA lipid particle (SMALP) system and its impacts including three dimensional structures and biochemical functions of peripheral and transmembrane proteins. Polymers systems are emerging to tackle the remaining challenges for wider use and future applications including in membrane proteomics, structural biology of transient or unstable states, and discovery of ligand and drug-like molecules specific for native lipid-bound states.     

Complexation of integral membrane proteins by phosphorylcholine-based amphipols

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b(6)f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols ( approximately 22 kDa) of low dispersity in length, containing approximately 30 mol% octyl side groups, approximately 35 mol% PC-groups, and approximately 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH> or =5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.     

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.     

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.     

Solution Behavior and Crystallization of Cytochrome bc 1 in the Presence of Amphipols

Detergents classically are used to keep membrane proteins soluble in aqueous solutions, but they tend to destabilize them. This problem can be largely alleviated thanks to the use of amphipols (APols), small amphipathic polymers designed to substitute for detergents. APols adsorb at the surface of the transmembrane region of membrane proteins, keeping them water-soluble while stabilizing them bio-chemically. Membrane protein/APol complexes have proven, however, difficult to crystallize. In this study, the composition and solution properties of complexes formed between mitochondrial cytochrome bc ₁ and A8-35, the most extensively used APol to date, have been studied by means of size exclusion chromatography, sucrose gradient sedimentation, and small-angle neutron scattering. Stable, monodisperse preparations of bc ₁/A8-35 complexes can be obtained, which, depending on the medium, undergo either repulsive or attractive interactions. Under crystallization conditions, diffracting three-dimensional crystals of A8-35-stabilized cytochrome bc ₁ formed, but only in the concomitant presence of APol and detergent.     

Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4–7 Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more “native” like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.     

Integrative systems and synthetic biology of cell-matrix adhesion sites

ABSTRACT

The complexity of cell-matrix adhesion convolves its roles in the development and functioning of multicellular organisms and their evolutionary tinkering. Cell-matrix adhesion is mediated by sites along the plasma membrane that anchor the actin cytoskeleton to the matrix via a large number of proteins, collectively called the integrin adhesome. Fundamental challenges for understanding how cell-matrix adhesion sites assemble and function arise from their multi-functionality, rapid dynamics, large number of components and molecular diversity. Systems biology faces these challenges in its strive to understand how the integrin adhesome gives rise to functional adhesion sites. Synthetic biology enables engineering intracellular modules and circuits with properties of interest. In this review I discuss some of the fundamental questions in systems biology of cell-matrix adhesion and how synthetic biology can help addressing them.     

Complexation of integral membrane proteins by phosphorylcholine-based amphipols

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b₆f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols (<M>≈ 22 kDa) of low dispersity in length, containing ∼ 30 mol% octyl side groups, ∼ 35 mol% PC-groups, and ∼ 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH ≥ 5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.     

Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy

Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank. Solid-state Nuclear Magnetic Resonance (SSNMR) has long been considered as an attractive alternative because it allows for studies of membrane proteins in both native-like membranes composed of synthetic lipids and in cell membranes. Over the past decade, SSNMR has been rapidly developing into a major structural method, and a growing number of membrane protein structures obtained by this technique highlights its potential. Here we discuss membrane protein sample requirements, review recent progress in SSNMR methodologies, and describe recent advances in characterizing membrane proteins in the environment of a cellular membrane.     

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for “artificial alpha repeat protein”) have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal “Velcro” to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.     

In situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein-tethering activities

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum via the specific interaction of the FFAT motif with endoplasmic reticulum–resident vesicle-associated membrane protein–associated proteins. This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins and of a protein construct derived from steroidogenic acute regulatory protein–related lipid transfer domain-3. Collectively, these data indicate that the creation of in situ artificial contact sites represents an efficient approach for studying the membrane-tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.     

The chemical biology of Ras lipidation

In this review, we summarize the successful interplay between three disciplines, organic synthesis, biophysics and cell biology, in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in molecular detail. Highlighting the example of the Ras proteins, we show how the development of new synthetic methodologies paved the road to the synthesis of lipidated peptides and--by a combination of chemical and molecular biological techniques--lipidated proteins as molecular tools. We further give an overview of the results of the biophysical properties and biological activities of the molecules synthesized by means of this interdisciplinary approach. This successful combination of different disciplines led to a better understanding of the selective targeting of Ras and related lipoproteins to the plasma membrane.     

Folding of diphtheria toxin T-domain in the presence of amphipols and fluorinated surfactants: Toward thermodynamic measurements of membrane protein folding

Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which, however, tend to destabilize them. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homopolymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of leakage of vesicle content indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. This article is part of a Special Issue entitled: Protein Folding in Membranes.