Stability Of Membrane Proteins Research Articles

Lipidic liquid crystalline cubic phases for preparation of ATP-hydrolysing enzyme electrodes

The lipidic liquid-crystalline cubic phase (LCP) is a membrane-mimetic material useful for the stabilization and structural analysis of membrane proteins. Here, we focused on the incorporation of the membrane ATP-hydrolysing sodium/potassium transporter Na+/K+-ATPase (NKA) into a monoolein-derived LCP. Small-angle X-ray scattering was employed for the determination of the LCP structure, which was of Pn3m symmetry for all the formulations studied. The fully characterized NKA-LCP material was immobilized onto a glassy carbon electrode, forming a highly stable enzyme electrode and a novel sensing platform. A typical NKA voltammetric signature was monitored via the anodic reaction of tyrosine and tryptophan residues. The in situ enzyme activity evaluation was based on the ability of NKA to transform ATP to ADP and free phosphate, the latter reacting with ammonium molybdate to form the ammonium phosphomolybdate complex under acidic conditions. The square-wave voltammetric detection of phosphomolybdate was performed and complemented with spectrophotometric measurement at 710nm. The anodic voltammetric response, corresponding to the catalytic ATP-hydrolysing function of NKA incorporated into the LCP, was monitored at around + 0.2V vs. Ag/AgCl in the presence or absence of ouabain, a specific NKA inhibitor. NKA incorporated into the LCP retained its ATP-hydrolysing activity for 7 days, while the solubilized protein became practically inactive. The novelty of this work is the first incorporation of NKA into a lipidic cubic phase with consequent enzyme functionality and stability evaluation using voltammetric detection. The application of LCPs could also be important in the further development of new membrane protein electrochemical sensors and enzyme electrodes.

Kinetic stability of membrane proteins.

Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

Lipidic Cubic Phase-Induced Membrane Protein Crystallization: Interplay Between Lipid Molecular Structure, Mesophase Structure and Properties, and Crystallogenesis

Obtaining well-diffracting crystals of membrane proteins, which is crucial to the molecular-level understanding of their intrinsic three-dimensional structure, dynamics, and function, represents a fundamental bottleneck in the field of structural biology. One of the major advances in the field of membrane protein structural determination was the realization that the nanostructured lipidic cubic phase (LCP) environment constitutes a membrane mimetic matrix that promotes solubilization, stabilization, and crystallization of specific membrane proteins. Despite two decades passing since the introduction of LCP-based membrane protein crystallization, the research community’s understanding of the processes that drive protein nucleation and macromolecular assembly in the LCP generally remains limited. In the current study, we present a detailed, systematic investigation into the relationship between the chemical structure of the lipid, the physical properties of the ensuing mesophase, the translational diffusion...

Inhibition of insulin/PI3K/AKT signaling decreases adipose Sortilin 1 in mice and 3 T3-L1 adipocytes

Sortilin 1(Sort1) is a vesicle trafficking receptor that mediates protein sorting in the endocytic and exocytic pathways. Sort1 is a component of the GLUT4 storage vesicles in adipocytes and is also involved in the regulation of adipogenesis. Sort1 protein is reduced in adipose of obese mice and humans, but the underlying cause is not fully understood. Here we report that insulin/PI3K/AKT signaling cascade critically regulates adipose Sort1 protein abundance. Administration of a PI3K inhibitor rapidly decreased Sort1 protein but not mRNA in adipose of chow-fed mice. In 3T3-L1 adipocytes, serum-starvation or inhibition of the PI3K/AKT signaling also decreased Sort1 protein without affecting Sort1 mRNA expression. Sort1 protein downregulation upon PI3K inhibition was blocked by pretreatment of MG132 but not Bafilomycin A1, suggesting that PI3K inhibition caused Sort1 degradation via the proteasome pathway. Using a phospho-specific Sort1 antibody, we showed that endogenous Sort1 was phosphorylated at S825 adjacent to the DXXLL sorting motif on the cytoplasmic tail. We demonstrated that mutagenesis that abolished Sort1 S825 phosphorylation decreased insulin-stimulated Sort1 localization on the plasma membrane and Sort1 protein stability in 3T3-L1 adipocytes. However, endogenous Sort1 phosphorylation at S825 was not affected by insulin stimulation or by inhibition of PI3K. In conclusion, this study revealed an important role of insulin signaling in regulating adipose Sort1 protein stability, and further suggests that impaired insulin signaling may underlie reduced adipose Sort1 in obesity. The cellular events downstream of insulin/PI3K/AKT signaling that mediates insulin regulation of Sort1 stability requires further investigation.

A Novel Interacting Protein SERP1 Regulates the N-Linked Glycosylation and Function of GLP-1 Receptor in the Liver.

Glucagon-like peptide 1 (GLP-1) exerts multiple effects on metabolism through its receptor, GLP-1R, in the liver. Activation and transduction of GLP-1R require complex interactions of largely unknown accessory proteins, and these processes are crucial to the response to endoplasmic reticulum (ER) stress. Using the membrane-based split ubiquitin yeast two-hybrid system (MYTH) and a human liver cDNA library, we obtained the human GLP-1R interactome and identified SERP1 as a potential interacting protein based on its ability to stabilize membrane proteins and facilitate N-linked glycosylation. GLP-1R and SERP1 were co-expressed in HEK-293 cells, and their interaction was confirmed by co-immunoprecipitation. We then found that overexpression of SERP1 could rescue GLP-1R glycosylation after application of tunicamycin to block N-linked glycosylation. SERP1 overexpression also attenuated exendin-4-stimulated cAMP accumulation and AMPK activation. However, the glycosylation and function of mutant GLP-1R, in which all three sites for N-linked glycosylation were mutated, were not increased with overexpression of SERP1. Moreover, as a GLP-1R interactor, SERP1 could also partly reverse the accumulation of tunicamycin-induced ER stress. Taken together, our findings identify a group of proteins that interact with GLP-1R and show that one specific interacting protein, SERP1, has an important role in facilitating the glycosylation of GLP-1R and rescuing its activities after ER stress induced by tunicamycin. J. Cell. Biochem. 118: 3616-3626, 2017. © 2017 Wiley Periodicals, Inc.

Lipid-like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies.

A crucial bottleneck in membrane protein structural biology is the difficulty in identifying a detergent that can maintain the stability and functionality of integral membrane proteins (IMPs). Detergents are poor membrane mimics, and their common use in membrane protein crystallography may be one reason for the challenges in obtaining high‐resolution crystal structures of many IMP families. Lipid‐like peptides (LLPs) have detergent‐like properties and have been proposed as alternatives for the solubilization of G protein‐coupled receptors and other membrane proteins. Here, we systematically analyzed the stabilizing effect of LLPs on integral membrane proteins of different families. We found that LLPs could significantly stabilize detergent‐solubilized IMPs in vitro. This stabilizing effect depended on the chemical nature of the LLP and the intrinsic stability of a particular IMP in the detergent. Our results suggest that screening a subset of LLPs is sufficient to stabilize a particular IMP, which can have a substantial impact on the crystallization and quality of the crystal.

Analysis and prediction of protein folding energy changes upon mutation by element specific persistent homology.

Site directed mutagenesis is widely used to understand the structure and function of biomolecules. Computational prediction of mutation impacts on protein stability offers a fast, economical and potentially accurate alternative to laboratory mutagenesis. Most existing methods rely on geometric descriptions, this work introduces a topology based approach to provide an entirely new representation of mutation induced protein stability changes that could not be obtained from conventional techniques. Topology based mutation predictor (T-MP) is introduced to dramatically reduce the geometric complexity and number of degrees of freedom of proteins, while element specific persistent homology is proposed to retain essential biological information. The present approach is found to outperform other existing methods in the predictions of globular protein stability changes upon mutation. A Pearson correlation coefficient of 0.82 with an RMSE of 0.92 kcal/mol is obtained on a test set of 350 mutation samples. For the prediction of membrane protein stability changes upon mutation, the proposed topological approach has a 84% higher Pearson correlation coefficient than the current state-of-the-art empirical methods, achieving a Pearson correlation of 0.57 and an RMSE of 1.09 kcal/mol in a 5-fold cross validation on a set of 223 membrane protein mutation samples. http://weilab.math.msu.edu/TML/TML-MP/. wei@math.msu.edu. Supplementary data are available at Bioinformatics online.

The non-bilayer lipid MGDG stabilizes the major light-harvesting complex (LHCII) against unfolding

In the photosynthetic apparatus of plants a high proportion of LHCII protein is needed to integrate 50% non-bilayer lipid MGDG into the lamellar thylakoid membrane, but whether and how the stability of the protein is also affected is not known. Here we use single-molecule force spectroscopy to map the stability of LHCII against mechanical unfolding along the polypeptide chain as a function of oligomerization state and lipid composition. Comparing unfolding forces between monomeric and trimeric LHCII demonstrates that the stability does not increase significantly upon trimerization but can mainly be correlated with specific contact sites between adjacent monomers. In contrast, unfolding of trimeric complexes in membranes composed of different thylakoid lipids reveals that the non-bilayer lipid MGDG substantially increases the mechanical stability of LHCII in many segments of the protein compared to other lipids such as DGDG or POPG. We attribute these findings to steric matching of conically formed MGDG and the hourglass shape of trimeric LHCII, thereby extending the role of non-bilayer lipids to the structural stabilization of membrane proteins in addition to the modulation of their folding, conformation and function.

Energetics of side-chain partitioning of β-signal residues in unassisted folding of a transmembrane β-barrel protein

The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane β-barrels form the β-signal motif required for assisted β-barrel assembly in vivo but are believed to be less important for β-barrel assembly in vitro. Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the β-signal motif to the unassisted folding of the model β-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the β-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain–specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP β-barrel scaffold. We conclude that the PagP C-terminal β-signal motif influences the folding cooperativity and stability of the folded β-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein β-signal motif depend on the nature of the amino acid side chain.

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.

Resorcinarene-Based Facial Glycosides: Implication of Detergent Flexibility on Membrane-Protein Stability.

As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from lipid environments and to maintain their solubility and stability in an aqueous medium. However, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of new agents with enhanced properties. In the current study, two classes of new amphiphiles are introduced, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), for which the alkyl chains are facially segregated from the carbohydrate head groups. Of these facial amphiphiles, two RGAs (RGA-C11 and RGA-C13) conferred markedly enhanced stability to four tested membrane proteins compared to a gold-standard conventional detergent. The relatively high water solubility and micellar stability of the RGAs compared to the RMAs, along with their generally favourable behaviours for membrane protein stabilisation described here, are likely to be, at least in part, a result of the high conformational flexibility of these glucosides. This study suggests that flexibility could be an important factor in determining the suitability of new detergents for membrane protein studies.

Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study: Implications of Detergent Kink for Micellar Properties.

Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties. Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n-dodecyl-β-d-maltoside (DDM) for all membrane proteins tested. Efficacy of the individual NBMs varied depending on the overall detergent shape and alkyl chain length. Specifically, NBMs with no kink in the lipophilic region conferred greater stability to the proteins than NBMs with a kink. In addition, long alkyl chain NBMs were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes novel maltoside detergents with enhanced protein-stabilizing properties but also suggests that overall detergent geometry has an important role in determining membrane protein stability. Notably, this is the first systematic study on the effect of detergent kinking on micellar properties and associated membrane protein stability.

Production of Isolated Giant Unilamellar Vesicles under High Salt Concentrations.

The cell membrane forms a dynamic and complex barrier between the living cell and its environment. However, its in vivo studies are difficult because it consists of a high variety of lipids and proteins and is continuously reorganized by the cell. Therefore, membrane model systems with precisely controlled composition are used to investigate fundamental interactions of membrane components under well-defined conditions. Giant unilamellar vesicles (GUVs) offer a powerful model system for the cell membrane, but many previous studies have been performed in unphysiologically low ionic strength solutions which might lead to altered membrane properties, protein stability and lipid-protein interaction. In the present work, we give an overview of the existing methods for GUV production and present our efforts on forming single, free floating vesicles up to several tens of μm in diameter and at high yield in various buffer solutions with physiological ionic strength and pH.

Durable vesicles for reconstitution of membrane proteins in biotechnology.

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with.

Side Chain Hydrophobicity Scale using the Tilted Beta-Barrel Protein PagP

Membrane proteins fold via the transfer of amino acid side chains from aqueous solvent into a lipid bilayer. Thus, knowing the free energy change of side chain transfer from water to bilayer will contribute to understanding structure, function and energetics of membrane proteins. We have measured the transfer free energies for all twenty amino acids using the scaffold protein PagP in a DLPC bilayer. Compared to previously measured transfer free energies, the overall trend agrees: hydrophobic residues are favorable and hydrophilic residues are unfavorable in the bilayer. However, we have found that the transfer of some polar the side chains are more unfavorable than previously observed. We hypothesize that the physical basis for these new findings may be the tilt of PagP with respect to the membrane. These results indicate membrane protein stability is governed not only by side chain desolvation, but also structural features such as depth and angle in the membrane.

Evaluating Bilayer Mechanical Properties in Protein Reconstituted GUVs

Biological membranes are complex systems where membrane proteins are surrounded by a bilayer composed of different types of lipids, with membranes from different organisms and organelles varying greatly in their composition and therefore physical properties. The mix of lipids in the membrane has additionally been shown to influence the activity, stability and conformation of many integral membrane proteins. In order to investigate the effect of incorporating protein on the mechanical properties of lipid bilayers we have reconstituted the Escherichia coli transporter lactose permease (LacY), a transmembrane protein representative of the major facilitator superfamily, into synthetic lipid vesicles. We observed directly the fluctuation of the lipid bilayer in these protein reconstituted GUVs, with sizes in the order of micrometers, by phase contrast microscopy. This allowed the determination of the bending rigidity, which characterizes the ability of membranes to bend under low stress, by fluctuation analysis. Changes in the bending rigidity parameter allowed us to get better insights into the effect of lipids and protein on the mechanical properties of GUVs in a quantitative fashion.

The role of interfacial lipids in stabilizing membrane protein oligomers.

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na+/H+ antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study.

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human β2 adrenergic receptor (β2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of β2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter.

Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.

Mesitylene‐Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane proteins compared to both DDM and the XMAs. This favourable behaviour is due likely to the increased hydrophobic density provided by the extra alkyl chain. Thus, this study not only describes new glucoside agents with potential for membrane protein research, but also introduces a new detergent design principle for future development.