Foldable Detergents for Membrane Protein Study: Importance of Detergent Core Flexibility in Protein Stabilization.

Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.

Diastereomeric Cyclopentane-Based Maltosides (CPMs) as Tools for Membrane Protein Study.

Amphiphilic agents, called detergents, are invaluable tools for studying membrane proteins. However, membrane proteins encapsulated by conventional head-to-tail detergents tend to denature or aggregate, necessitating the development of structurally distinct molecules with improved efficacy. Here, a novel class of diastereomeric detergents with a cyclopentane core unit, designated cyclopentane-based maltosides (CPMs), were prepared and evaluated for their ability to solubilize and stabilize several model membrane proteins. A couple of CPMs displayed enhanced behavior compared with the benchmark conventional detergent, n-dodecyl-ß-d-maltoside (DDM), for all the tested membrane proteins including two G-protein-coupled receptors (GPCRs). Furthermore, CPM-C12 was notable for its ability to confer enhanced membrane protein stability compared with the previously developed conformationally rigid NBMs [J. Am. Chem. Soc. 2017, 139, 3072] and LMNG. The effect of the individual CPMs on protein stability varied depending on both the detergent configuration (cis/trans) and alkyl chain length, allowing us draw conclusions on the detergent structure-property-efficacy relationship. Thus, this study not only provides novel detergent tools useful for membrane protein research but also reports on structural features of the detergents critical for detergent efficacy in stabilizing membrane proteins.

Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability.

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-ß-d-maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human ß2 adrenergic receptor (ß2 AR), a G-protein coupled receptor, and its complex with Gs protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.

Trehalose-cored amphiphiles for membrane protein stabilization: importance of the detergent micelle size in GPCR stability.

Despite their importance in biology and medicinal chemistry, structural and functional studies of membrane proteins present major challenges. To study diverse membrane proteins, it is crucial to have the correct detergent to efficiently extract and stabilize the proteins from the native membranes for biochemical/biophysical downstream analyses. But many membrane proteins, particularly eukaryotic ones, are recalcitrant to stabilization and/or crystallization with currently available detergents and thus there are major efforts to develop novel detergents with enhanced properties. Here, a novel class of trehalose-cored amphiphiles are introduced, with multiple alkyl chains and carbohydrates projecting from the trehalose core unit are introduced. A few members displayed enhanced protein stabilization behavior compared to the benchmark conventional detergent, n-dodecyl-ß-d-maltoside (DDM), for multiple tested membrane proteins: (i) a bacterial leucine transporter (LeuT), (ii) the R. capsulatus photosynthetic superassembly, and (iii) the human ß2 adrenergic receptor (ß2AR). Due to synthetic convenience and their favourable behaviors for a range of membrane proteins, these agents have potential for membrane protein research. In addition, the detergent property-efficacy relationship discussed here will guide future design of novel detergents.

Correction: Trehalose-cored amphiphiles for membrane protein stabilization: importance of the detergent micelle size in GPCR stability.

Correction for 'Trehalose-cored amphiphiles for membrane protein stabilization: importance of the detergent micelle size in GPCR stability' by Manabendra Das et al., Org. Biomol. Chem., 2019, 17, 3249-3257.

Rationally Engineered Tandem Facial Amphiphiles for Improved Membrane Protein Stabilization Efficacy.

A new family of tandem facial glucosides/maltosides (TFGs/TFMs) for membrane protein manipulation was prepared. The best detergent varied depending on the hydrophobic thickness of the target protein, but ether-based TFMs (TFM-C0E, TFM-C3E, and TFM-C5E) were notable for their ability to confer higher membrane protein stability than the previously developed amide-based TFA-1 (P. S. Chae, K. Gotfryd, J. Pacyna, L. J. W. Miercke, S. G. F. Rasmussen, R. A. Robbins, R. R. Rana, C. J. Loland, B. Kobilka, R. Stroud, B. Byrne, U. Gether, S. H. Gellman, J. Am. Chem. Soc. 2010, 132, 16750-16752). Thus, this study not only introduces novel agents with the potential to be used in membrane protein research but also highlights the importance of both the hydrophobic length and linker functionality of the detergent in stabilizing membrane proteins.

Steroid-Based Amphiphiles for Membrane Protein Study: The Importance of Alkyl Spacers for Protein Stability.

Membrane proteins allow effective communication between cells and organelles and their external environments. Maintaining membrane protein stability in a non-native environment is the major bottleneck to their structural study. Detergents are widely used to extract membrane proteins from the membrane and to keep the extracted protein in a stable state for downstream characterisation. In this study, three sets of steroid-based amphiphiles-glyco-diosgenin analogues (GDNs) and steroid-based pentasaccharides either lacking a linker (SPSs) or containing a linker (SPS-Ls)-have been developed as new chemical tools for membrane protein research. These detergents were tested with three membrane proteins in order to characterise their ability to extract membrane proteins from the membrane and to stabilise membrane proteins long-term. Some of the detergents, particularly the SPS-Ls, displayed favourable behaviour with the tested membrane proteins. This result indicates the potential utility of these detergents as chemical tools for membrane protein structural study and a critical role of the simple alkyl spacer in determining detergent efficacy.

An Engineered Lithocholate-Based Facial Amphiphile Stabilizes Membrane Proteins: Assessing the Impact of Detergent Customizability on Protein Stability.

Amphiphiles are critical tools for the structural and functional study of membrane proteins. Membrane proteins encapsulated by conventional head-to-tail detergents tend to undergo structural degradation, necessitating the development of structurally novel agents with improved efficacy. In recent years, facial amphiphiles have yielded encouraging results in terms of membrane protein stability. Herein, we report a new facial detergent (i.e., LFA-C4) that confers greater stability to tested membrane proteins than the bola form analogue. Owing to the increased facial property and the adaptability of the detergent micelles in complex with different membrane proteins, LFA-C4 yields increased stability compared to n-dodecyl-ß-d-maltoside (DDM). Thus, this study not only describes a novel maltoside detergent with enhanced protein-stabilizing properties, but also shows that the customizable nature of a detergent plays an important role in the stabilization of membrane proteins. Owing to both synthetic convenience and enhanced stabilization efficacy for a range of membrane proteins, the new agent has major potential in membrane protein research.

A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability.

The study of membrane proteins is extremely challenging, mainly because of the incompatibility of the hydrophobic surfaces of membrane proteins with an aqueous medium. Detergents are essential agents used to maintain membrane protein stability in non-native environments. However, conventional detergents fail to stabilize the native structures of many membrane proteins. Development of new amphipathic agents with enhanced efficacy for membrane protein stabilization is necessary to address this important problem. We have designed and synthesized linear and branched mannitol-based amphiphiles (MNAs), and comparative studies showed that most of the branched MNAs had advantages over the linear agents in terms of membrane protein stability. In addition, a couple of the new MNAs displayed favorable behaviors compared to n-dodecyl-ß-d-maltoside and the previously developed MNAs in maintaining the native protein structures, indicating potential utility of these new agents in membrane protein study.

Vitamin E-based glycoside amphiphiles for membrane protein structural studies.

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile-lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.

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-ß-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.

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.

New penta-saccharide-bearing tripod amphiphiles for membrane protein structure studies.

Integral membrane proteins either alone or as complexes carry out a range of key cellular functions. Detergents are indispensable tools in the isolation of membrane proteins from biological membranes for downstream studies. Although a large number of techniques and tools, including a wide variety of detergents, are available, purification and structural characterization of many membrane proteins remain challenging. In the current study, a new class of tripod amphiphiles bearing two different penta-saccharide head groups, designated TPSs, were developed and evaluated for their ability to extract and stabilize a range of diverse membrane proteins. Variations in the structures of the detergent head and tail groups allowed us to prepare three sets of the novel agents with distinctive structures. Some TPSs (TPS-A8 and TPS-E7) were efficient at extracting two proteins in a functional state while others (TPS-E8 and TPS-E10L) conferred marked stability to all membrane proteins (and membrane protein complexes) tested here compared to a conventional detergent. Use of TPS-E10L led to clear visualization of a receptor-Gs complex using electron microscopy, indicating profound potential in membrane protein research.

Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.

Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies.

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

Isomeric Detergent Comparison for Membrane Protein Stability: Importance of Inter-Alkyl-Chain Distance and Alkyl Chain Length.

Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile-lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins.

Accessible Mannitol-Based Amphiphiles (MNAs) for Membrane Protein Solubilisation and Stabilisation.

Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the ß2 adrenergic receptor (ß2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the ß2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.

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.

3,4-Bis(hydroxymethyl)hexane-1,6-diol-based Maltosides (HDMs) for Membrane-Protein Study: Importance of Detergent Rigidity-Flexibility Balance in Protein Stability.

Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be particularly poor, necessitating development of novel detergents with improved properties. Here, we prepared a novel class of detergents, designated 3,4-bis(hydroxymethyl)hexane-1,6-diol-based maltosides (HDMs). When tested on three membrane proteins, including two G-protein-coupled receptors (GPCRs), the new detergents displayed significantly better behaviors compared with DDM. Moreover, the HDMs were superior or comparable to LMNG, an amphiphile widely used for GPCR structural study. An optimal balance of detergent rigidity vs. flexibility of the HDMs is likely responsible for their favorable behaviors toward membrane-protein stability. Thus, the current study not only introduces the HDMs, with significant potential for membrane-protein structural study, but also suggests a useful guideline for designing novel detergents for membrane-protein research.

Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltose-tris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability.

Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.