High-resolution Structures Of Membrane Proteins Research Articles

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Elucidating the Structural Dynamics of Integrin α IIbβ 3 from Native Platelet Membranes By Cryo-EM Coupled with Build and Retrieve Data Processing Methodology

Background: Platelets play a critical role in physiological processes by sensing the extracellular environment through their membrane proteins. Changing the quantity or quality of these proteins can profoundly affect platelet physiology and impact health. The native membrane environment provides essential additional regulatory cues that impact the protein structure and mechanism of action. Single-particle cryogenic electron microscopy (cryo-EM) has transformed structural biology by allowing high-resolution structures of membrane proteins and large macromolecules to be solved from highly purified, homogeneous samples. Our recent breakthroughs in data processing make it feasible to obtain high-resolution protein structures from crude preparations in their native environments by integrating cryo-EM with the novel “Build and Retrieve” (BaR) data processing methodology. By performing in silico purification, image sorting, and model building from large heterogenous datasets, this iterative bottom-up methodology opens new avenues for an in-depth systems biology approach with structural biology that will uncover how the native environment, including lipid and metal binding, post-translational modifications, and co-factor associations regulate platelet function at the molecular level. Method: Whole blood was collected, and the platelet membrane proteins were solubilized by 1% n-Dodecyl-β-D-Maltoside (DDM) and incorporated into lipidic nanodisc MSP1E3D1 directly from the endogenous source. After being enriched by size exclusion chromatography, the mixture of proteins at ~200 kDa was directly applied on cryo-EM grids. Data were collected on Titan Krios G3i cryogenic transmission electron microscope equipped with a BioQuantum K3 camera. All data processing was done in cryoSPARC using the BaR protocol. The near-atomic-resolution cryo-EM maps were used for protein identification using the program phenix.sequence_from_map in the PHENIX suite. The final protein models were built by Coot and refined by PHENIX. Results: We have used using cryo-EM followed by the BaR method to solve the first unmodified integrin α IIbβ 3 structure directly from resting human platelet membranes in its inactivated and intermediate states at 2.76Å and 2.49Å, respectively. From these two structures, we observed the ion binding sites at the β1-domain and β-propeller domain in agreement with previous “classical” α IIbβ 3 structural studies using a purified sample. More importantly, we were able to assign endogenous N-linked glycan sites, which reflected how glycosylation naturally regulates α IIbβ 3 dynamic and function on resting human platelets. Finally, we also solved a novel third unique dimer conformation of α IIbβ 3 at 2.61Å formed by two intermediate-states of α IIbβ 3. This may indicate a previously unknown self-regulatory mechanism of α IIbβ 3 in its native environment. Conclusion: In conclusion, our data show the power of using cryo-EM with the BaR method to uncover novel structures directly from natural sources to identify unrecognized regulation mechanisms for proteins without artifacts due to purification processes. A distinct advantage of the BaR method is that it is an unbiased approach for solving multiple structures from raw samples, which highlights the potential of building a protein structural atlas from platelets. These data have the potential to enrich our understanding of platelet signaling circuitry and foster the development of higher-quality diagnoses and treatments for patients with platelet disorders.

Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.

Role of surfactants in electron cryo-microscopy film preparation

Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However, obtaining cryo-EM grids of sufficient quality for high-resolution structural analysis remains a major bottleneck. One of the difficulties arises from the presence of detergents, which often leads to a lack of control of the ice thickness. Amphipathic polymers such as amphipols (APols) are detergent substitutes, which have proven to be valuable tools for cryo-EM studies. In this work, we investigate the physico-chemical behavior of APol- and detergent-containing solutions and show a correlation with the properties of vitreous thin films in cryo-EM grids. This study provides new insight on the potential of APols, allowing a better control of ice thickness while limiting protein adsorption at the air-water interface, as shown with the full-length mouse serotonin 5-HT3A receptor whose structure has been solved in APol. These findings may speed up the process of grid optimization to obtain high-resolution structures of membrane proteins.

Tris(hydroxymethyl)aminomethane Linker-Bearing Triazine-Based Triglucosides for Solubilization and Stabilization of Membrane Proteins.

High-resolution membrane protein structures are essential for a fundamental understanding of the molecular basis of diverse cellular processes and for drug discovery. Detergents are widely used to extract membrane-spanning proteins from membranes and maintain them in a functional state for downstream characterization. Due to limited long-term stability of membrane proteins encapsulated in conventional detergents, development of novel agents is required to facilitate membrane protein structural study. In the current study, we designed and synthesized tris(hydroxymethyl)aminomethane linker-bearing triazine-based triglucosides (TTGs) for solubilization and stabilization of membrane proteins. When these glucoside detergents were evaluated for four membrane proteins including two G protein-coupled receptors, a few TTGs including TTG-C10 and TTG-C11 displayed markedly enhanced behaviors toward membrane protein stability relative to two maltoside detergents [DDM (n-dodecyl-β-d-maltoside) and LMNG (lauryl maltose neopentyl glycol)]. This is a notable feature of the TTGs as glucoside detergents tend to be inferior to maltoside detergents at stabilizing membrane proteins. The favorable behavior of the TTGs for membrane protein stability is likely due to the high hydrophobicity of the lipophilic groups, an optimal range of hydrophilic-lipophilic balance, and the absence of cis-trans isomerism.

Cryo-electron Microscopic Analysis of Single-Pass Transmembrane Receptors.

Single-pass transmembrane receptors (SPTMRs) represent a diverse group of integral membrane proteins that are involved in many essential cellular processes, including signal transduction, cell adhesion, and transmembrane transport of materials. Dysregulation of the SPTMRs is linked with many human diseases. Despite extensive efforts in past decades, the mechanisms of action of the SPTMRs remain incompletely understood. One major hurdle is the lack of structures of the full-length SPTMRs in different functional states. Such structural information is difficult to obtain by traditional structural biology methods such as X-ray crystallography and nuclear magnetic resonance (NMR). The recent rapid development of single-particle cryo-electron microscopy (cryo-EM) has led to an exponential surge in the number of high-resolution structures of integral membrane proteins, including SPTMRs. Cryo-EM structures of SPTMRs solved in the past few years have tremendously improved our understanding of how SPTMRs function. In this review, we will highlight these progresses in the structural studies of SPTMRs by single-particle cryo-EM, analyze important structural details of each protein involved, and discuss their implications on the underlying mechanisms. Finally, we also briefly discuss remaining challenges and exciting opportunities in the field.

Advances in mass spectrometry-based footprinting of membrane proteins.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo-electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS-based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II-transducer complex.

Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII-NpHtrII) was performed. The influence of the polydispersity of NpSRII-NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

Correlating ion channel structure and function.

Recent developments in cryogenic electron microscopy (cryo-EM) led to an exponential increase in high-resolution structures of membrane proteins, and in particular ion channels. However, structures alone can only provide limited information about the workings of these proteins. In order to understand ion channel function and regulation in molecular detail, the obtained structural data need to be correlated to functional states of the same protein. Here, we describe several techniques that can be employed to study ion channel structure and function in vitro and under defined, similar conditions. Lipid nanodiscs provide a native-like environment for membrane proteins and have become a valuable tool in membrane protein structural biology and biophysics. Combined with liposome-based flux assays for the kinetic analysis of ion channel activity as well as electrophysiological recordings, researchers now have access to an array of experimental techniques allowing for detailed structure-function correlations using purified components. Two examples are presented where we put emphasis on the lipid environment and time-resolved techniques together with mutations and protein engineering to interpret structural data obtained from single particle cryo-EM on cyclic nucleotide-gated or Ca2+-gated K+ channels. Furthermore, we provide short protocols for all the assays used in our work so that others can adapt these techniques to their experimental needs. Comprehensive structure-function correlations are essential in order to pharmacologically target channelopathies.

A guide to membrane protein X-ray crystallography.

Membrane proteins play critical physiological roles in all organisms, from ion transport and signal transduction to multidrug resistance. Elucidating their 3D structures is essential for understanding their functions, and this information can also be exploited for structure-aided drug discovery efforts. In this regard, X-ray crystallography has been the most widely used technique for determining the high-resolution 3D structures of membrane proteins. However, the success of this technique is dependent on efficient protein extraction, solubilization, stabilization, and generating diffracting crystals. Each of these steps can impose great challenges for membrane protein crystallographers. In this review, the process of generating membrane protein crystals from protein extraction and solubilization to structure determination is discussed. In addition, the current methods for precrystallization screening and a few strategies to increase the chance of crystallizing challenging membrane proteins are introduced.

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, “footprinting” studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver.

Be Cautious with Crystal Structures of Membrane Proteins or Complexes Prepared in Detergents.

Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM — an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed.

Raman Scattering: From Structural Biology to Medical Applications

This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.

Voltage-Dependent Profile Structures of a Kv-Channel via Time-Resolved Neutron Interferometry

Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments.

Emerging Diversity in Lipid-Protein Interactions.

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid–protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid–protein interactions, and to link lipid–protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid–protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid–protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid–protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid–protein interactions.

Combining Structural Data with Computational Methodologies to Investigate Structure-Function Relationships in TRP Channels.

Since the emergence of high-resolution three-dimensional structures of membrane proteins, and the increasing availability of state-of-the-art algorithms and high-performance-computing facilities, classical molecular dynamics (MD) simulations have become a routine device to explore the molecular behavior of these proteins. The rise of cryo-electron microscopy (cryo-EM) as a credible experimental tool to resolve structures at an atomic level has revolutionized structural biology in recent years, culminating in the disclosure of the first high-resolution three-dimensional structure of a transient receptor potential (TRP) channel, the vanilloid receptor 1 (TRPV1). As a result, the number of research articles investigating the molecular behavior of TRP channels using macromolecular simulation techniques has proliferated. This review provides an overview of the current state of this field, including our understanding of TRP channel structure, the framework of classical MD simulations, and how to perform such simulations to investigate structure-function relationships in TRP channels.

Fluorescence-based approaches for monitoring membrane receptor oligomerization

Membrane protein structures are highly under-represented relative to water-soluble protein structures in the protein databank. This is especially the case because membrane proteins represent more than 30% of proteins encoded in the human genome yet contribute to less than 10% of currently known structures (Torres et al. in Trends Biol Sci 28:137-144, 2003). Obtaining high-resolution structures of membrane proteins by traditional methods such as NMR and x-ray crystallography is challenging, because membrane proteins are difficult to solubilise, purify and crystallize. Consequently, development of methods to examine protein structure in situ is highly desirable. Fluorescence is highly sensitive to protein structure and dynamics (Lakowicz in Principles of fluorescence spectroscopy, Springer, New York, 2007). This is mainly because of the time a fluorescence probe molecule spends in the excited state. Judicious choice and placement of fluorescent molecule(s) within a protein(s) enables the experimentalist to obtain information at a specific site(s) in the protein (complex) of interest. Moreover, the inherent multi-dimensional nature of fluorescence signals across wavelength, orientation, space and time enables the design of experiments that give direct information on protein structure and dynamics in a biological setting. The purpose of this review is to introduce the reader to approaches to determine oligomeric state or quaternary structure at the cell membrane surface with the ultimate goal of linking the oligomeric state to the biological function. In the first section, we present a brief overview of available methods for determining oligomeric state and compare their advantages and disadvantages. In the second section, we highlight some of the methods developed in our laboratory to address contemporary questions in membrane protein oligomerization. In the third section, we outline our approach to determine the link between protein oligomerization and biological activity.

Cryo-electron microscopy of membrane proteins.

Membrane proteins represent a large proportion of the proteome, but have characteristics that are problematic for many methods in modern molecular biology (that have often been developed with soluble proteins in mind). For structural studies, low levels of expression and the presence of detergent have been thorns in the flesh of the membrane protein experimentalist. Here we discuss the use of cryo-electron microscopy in breakthrough studies of the structures of membrane proteins. This method can cope with relatively small quantities of sample and with the presence of detergent. Until recently, cryo-electron microscopy could not deliver high-resolution structures of membrane proteins, but recent developments in transmission electron microscope technology and in the image processing of single particles imaged in the microscope have revolutionized the field, allowing high resolution structures to be obtained. Here we focus on the specific issues surrounding the application of cryo-electron microscopy to the study of membrane proteins, especially in the choice of a system to keep the protein soluble.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

Tandem malonate-based glucosides (TMGs) for membrane protein structural studies

High-resolution membrane protein structures are essential for understanding the molecular basis of diverse biological events and important in drug development. Detergents are usually used to extract these bio-macromolecules from the membranes and maintain them in a soluble and stable state in aqueous solutions for downstream characterization. However, many eukaryotic membrane proteins solubilized in conventional detergents tend to undergo structural degradation, necessitating the development of new amphiphilic agents with enhanced properties. In this study, we designed and synthesized a novel class of glucoside amphiphiles, designated tandem malonate-based glucosides (TMGs). A few TMG agents proved effective at both stabilizing a range of membrane proteins and extracting proteins from the membrane environment. These favourable characteristics, along with synthetic convenience, indicate that these agents have potential in membrane protein research.