Stability Of Integral Membrane Proteins Research Articles

Effects of non-ionic and zwitterionic detergents on soluble proteins during native mass spectrometry experiments

Detergents are commonly employed for solubilization and stabilization of integral membrane proteins. Their effects on the charge states of detergent-solubilized membrane proteins in native mass spectrometry measurements have previously been described. These effects can be mediated through the transmembrane region directly interacting with the detergent micelle or through the soluble domains of the proteins getting in close contact with detergent micelles during electrospray ionization. Here, we explore the effects of detergent micelles on soluble proteins during native mass spectrometry aiming at distinguishing these two events. Specifically, we employ two non-ionic and one zwitterionic detergents as well as a variety of standard proteins differing in size, oligomeric state and surface hydrophobicity, and evaluate the observed charge states in the absence and presence of detergents. Using ion mobility-mass spectrometry, we assess the proteins' stability as well as the ability to maintain non-covalent interactions with ligands. Finally, we examine lipid transfer from mixed detergent-lipid micelles containing the various detergents to soluble proteins. In summary, we found that C8E4 detergent reduces the proteins’ charge states, stabilizes the proteins and facilitates lipid transfer.

Understanding phospholipid function: Why are there so many lipids?

In the 1970s, phospholipids were still considered mere building blocks of the membrane lipid bilayer, but the subsequent realization that phospholipids could also serve as second messengers brought new interest to the field. My own passion for the unique amphipathic properties of lipids led me to seek other, non-signaling functions for phospholipids, particularly in their interactions with membrane proteins. This seemed to be the last frontier in protein chemistry and enzymology to be conquered. I was fortunate to find my way to Eugene Kennedy's laboratory, where both membrane proteins and phospholipids were the foci of study, thus providing a jumping-off point for advancing our fundamental understanding of lipid synthesis, membrane protein biosynthesis, phospholipid and membrane protein trafficking, and the cellular roles of phospholipids. After purifying and characterizing enzymes of phospholipid biosynthesis in Escherichia coli and cloning of several of the genes encoding these enzymes in E. coli and Saccharomyces cerevisiae, I was in a position to alter phospholipid composition in a systematic manner during the cell cycle in these microorganisms. My group was able to establish, contrary to common assumption (derived from the fact that membrane proteins retain activity in detergent extracts) that phospholipid environment is a strong determining factor in the function of membrane proteins. We showed that molecular genetic alterations in membrane lipid composition result in many phenotypes, and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins. Here I present my personal "reflections" on how our understanding of phospholipid functions has evolved.

Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications.

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.

Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices.

The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analyzed via coarse grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations, we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of a family of helix sequences. We illustrate this software via analyses of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analyses of these ensembles of simulations, we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application, we use CGMD simulations to examine the self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase and analyze the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers a proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins.

Membrane Depth-Dependent Energetic Contribution of the Tryptophan Side Chain to the Stability of Integral Membrane Proteins

Lipid solvation provides the primary driving force for the insertion and folding of integral membrane proteins. Although the structure of the lipid bilayer is often simplified as a central hydrophobic core sandwiched between two hydrophilic interfacial regions, the complexity of the liquid-crystalline bilayer structure and the gradient of water molecules across the bilayer fine-tune the energetic contributions of individual amino acid residues to the stability of membrane proteins at different depths of the bilayer. The tryptophan side chain is particularly interesting because despite its widely recognized role in anchoring membrane proteins in lipid bilayers, there is little consensus about its hydrophobicity among various experimentally determined hydrophobicity scales. Here we investigated how lipid-facing tryptophan residues located at different depths in the bilayer contribute to the stability of integral membrane proteins using outer membrane protein A (OmpA) as a model. We replaced all lipid-contacting residues of the first transmembrane β-strand of OmpA with alanines and individually incorporated tryptophans in these positions along the strand. By measuring the thermodynamic stability of these proteins, we found that OmpA is slightly more stable when tryptophans are placed in the center of the bilayer and that it is somewhat destabilized as tryptophans approach the interfacial region. However, this trend may be partially reversed when a moderate concentration of urea rather than water is taken as the reference state. The measured stability profiles are driven by similar profiles of the m-value, a parameter that reflects the shielding of hydrophobic surface area from water. Our results indicate that knowledge of the free energy level of the protein's unfolded reference state is important for quantitatively assessing the stability of membrane proteins, which may explain differences in observed profiles between in vivo and in vitro scales.

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol − 1 to 23.4 ± 1.5 kcal mol − 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol − 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol − 1 to 10.9 ± 1.0 kcal mol − 1 , indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3–6 kcal mol − 1 . Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k u H2O, from ~1 × 10 − 16 s − 1 to ~1 × 10 − 4 s − 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol − 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein–retinal interactions that increase the rigidity and compactness of the polypeptide chain.

Trapping and Stabilization of Integral Membrane Proteins by Hydrophobically Grafted Glucose-Based Telomers

Amphipols (APols) are short amphipathic polymers designed to adsorb onto the transmembrane surface of membrane proteins, keeping them water-soluble in the absence of detergent. Current APols carry charged groups, which is a limitation for certain types of applications. This has prompted the development of totally nonionic amphiphols (NAPols). In a previous work, glucose-based NAPols synthesized by free-radical cotelomerization of hydrophilic and amphiphilic monomers proved to be able to keep membrane proteins soluble (Sharma et al. Langmuir 2008, 24, 13581-13590). This provided a proof of principle, but the cumbersome synthesis prevented large-scale production and any detailed biochemical studies. In the present work, we describe a new synthesis route for NAPols based on grafting alkyl chains onto a glucosylated homotelomer. The NAPols thus prepared are highly water soluble. In aqueous solutions, they assemble into small, homogeneous particles similar to those formed by ionic APols. Two model membrane proteins, bacteriorhodopsin and the transmembrane domain of OmpA, form with NAPols small, well-defined water-soluble complexes whose size is comparable to that observed with ionic APols. Complexation by NAPols strongly stabilizes bacteriorhodopsin against denaturation. Glucosylated NAPols thus appear as a promising alternative to ionic APols for such applications as ion-exchange chromatography, isoelectrofocusing, and, possibly, structural approaches such as NMR and crystallography.

Stability of Integral Membrane Proteins under High Hydrostatic Pressure: The LH2 and LH3 Antenna Pigment−Protein Complexes from Photosynthetic Bacteria

The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure, quite some of them remain apparently intact. The pigmented photosynthetic antenna complexes thus constitute a suitable model system for studying in detail the stability of integral membrane proteins.

Designing Facial Amphiphiles for the Stabilization of Integral Membrane Proteins

An added dimension: Amphiphiles with two-dimensional hydrophobic and hydrophilic faces represent a departure from traditional one-dimensional polar-head/nonpolar-tail molecules for the stabilization of integral membrane proteins. A prototypical example of this design has been synthesized from cholic acid (see picture), and was successfully used in the stabilization of two integral membrane proteins (represented by the blue/green cylinder).

Role of Aromatic Side Chains in the Folding and Thermodynamic Stability of Integral Membrane Proteins

Aromatic residues are frequently found in helical and beta-barrel integral membrane proteins enriched at the membrane-water interface. Although the importance of these residues in membrane protein folding has been rationalized by thermodynamic partition measurements using peptide model systems, their contribution to the stability of bona fide membrane proteins has never been demonstrated. Here, we have investigated the contribution of interfacial aromatic residues to the thermodynamic stability of the beta-barrel outer membrane protein OmpA from Escherichia coli in lipid bilayers by performing extensive mutagenesis and equilibrium folding experiments. Isolated interfacial tryptophanes contribute -2.0 kcal/mol, isolated interfacial tyrosines contribute -2.6 kcal/mol, and isolated interfacial phenylalanines contribute -1.0 kcal/mol to the stability of this protein. These values agree well with the prediction from the Wimley-White interfacial hydrophobicity scale, except for tyrosine residues, which contribute more than has been expected from the peptide models. Double mutant cycle analysis reveals that interactions between aromatic side chains become significant when their centroids are separated by less than 6 A but are nearly insignificant above 7 A. Aromatic-aromatic side chain interactions are on the order of -1.0 to -1.4 kcal/mol and do not appear to depend on the type of aromatic residue. These results suggest that the clustering of aromatic side chains at membrane interfaces provides an additional heretofore not yet recognized driving force for the folding and stability of integral membrane proteins.

Measurement of Thermodynamic Parameters for Hydrophobic Mismatch 2: Intermembrane Transfer of a Transmembrane Helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.

Stabilization of integral membrane proteins in aqueous solution using fluorinated surfactants

Surfactants carrying either a hydrocarbon or a fluorocarbon alkyl chain have been synthesized. The polar head was either tris(hydroxymethyl)acrylamidomethane (THAM), telomerized THAM, or a glycosylated THAM moiety. The aqueous solubility of some of these molecules was increased by oxidizing to a sulfoxide the thioether function that associates their hydrophobic and hydrophilic moieties. In all cases, the critical micellar concentration was principally determined by the length and chemical nature of the alkyl chain. The usefulness of these surfactants in handling integral membrane proteins in solution has been examined using as test materials chloroplast thylakoid membranes and the photosynthetic complex cytochrome b 6 f. In keeping with earlier observations in other systems, none of the fluorinated surfactants was able to solubilize thylakoid membranes. Transfer to a solution of fluorinated surfactant of b 6 f complexes that had been solubilized and purified in the presence of a classical detergent usually resulted in aggregation and precipitation of the protein, while most homologous molecules with hydrocarbon chains did keep the b 6 f complex soluble. Two of the fluorinated surfactants, however, proved able to maintain the b 6 f complex water-soluble, intact, and enzymatically active. Because of their limited affinity for lipid alkyl chains and other hydrocarbon surfaces, fluorinated surfactants appear as potentially interesting tools for the study of membrane proteins that do not stand well exposure to classical detergents.

Role of the actin cytoskeleton on epithelial Na+ channel regulation

The regulatory role of actin filament organization on epithelial Na+ channel activity is reviewed in this report. The actin cytoskeleton, consisting of actin filaments and associated actin-binding proteins, is essential to various cellular events including the maintenance of cell shape, the onset of cell motility, and the distribution and stability of integral membrane proteins. Functional interactions between the actin cytoskeleton and specific membrane transport proteins are, however, not as well understood. Recent studies from our laboratory have determined that dynamic changes in the actin cytoskeletal organization may represent a novel signaling mechanism in the regulation of ion transport in epithelia. This report summarizes work conducted in our laboratory leading to an understanding of the molecular steps associated with the regulatory role of the actin-based cytoskeleton on epithelial Na+ channel function. The basis of this interaction lies on the regulation by actin-binding proteins and adjacent structures, of actin filament organization which in turn, modulates ion channel activity. The scope of this interaction may extend to such relevant cellular events as the vasopressin response in the kidney.