Novel Xylene‐Linked Maltoside Amphiphiles (XMAs) for Membrane Protein Stabilisation

Membrane proteins are key functional players in biological systems. These biomacromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions. Conventional detergents are commonly used for membrane protein manipulation, but membrane proteins surrounded by these agents often undergo denaturation and aggregation. In this study, a novel class of maltoside‐bearing amphiphiles, with a xylene linker in the central region, designated xylene‐linked maltoside amphiphiles (XMAs) was developed. When these novel agents were evaluated with a number of membrane proteins, it was found that XMA‐4 and XMA‐5 have particularly favourable efficacy with respect to membrane protein stabilisation, indicating that these agents hold significant potential for membrane protein structural study.     

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.     

Improved Glucose‐Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization and Stabilization

Membrane proteins are inherently amphipathic and undergo dynamic conformational changes for proper function within native membranes. Maintaining the functional structures of these biomacromolecules in aqueous media is necessary for structural studies but difficult to achieve with currently available tools, thus necessitating the development of novel agents with favorable properties. This study introduces several new glucose‐neopentyl glycol (GNG) amphiphiles and reveals some agents that display favorable behaviors for the solubilization and stabilization of a large, multi‐subunit membrane protein assembly. Furthermore, a detergent structure–property relationship that could serve as a useful guideline for the design of novel amphiphiles is discussed.     

Novel Tripod Amphiphiles for Membrane Protein Analysis

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution. The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by the membrane proteins in their native forms and the polar solvent molecules. Here, we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to an enhanced protein solubilisation and stabilisation compared to both conventional detergents and previously described tripod amphiphiles.     

Hydrophobic Variations of N‐Oxide Amphiphiles for Membrane Protein Manipulation: Importance of Non‐hydrocarbon Groups in the Hydrophobic Portion

Amphipathic agents called detergents serve as membrane‐mimetic systems to maintain the native structures of membrane proteins during their manipulation. However, membrane proteins solubilized in conventional detergents tend to undergo denaturation and aggregation, necessitating the development of novel amphipathic agents with enhanced properties. Here we describe several new amphiphiles that contain an N‐oxide group as the hydrophilic portion. The new amphiphiles have been evaluated for the ability to solubilize and stabilize a fragile multi‐subunit assembly from biological membranes. We found that cholate‐based agents were promising in supporting retention of the native protein quaternary structure, while deoxycholate‐based amphiphiles were highly efficient in extracting/solubilizing the intact superassembly from the native membrane. Monitoring superassembly solubilization and stabilization as a function of variation in amphiphile structure led us to propose that a non‐hydrocarbon moiety such as an amide, ether, or a hydroxy group present in the lipophilic regions can manifest distinctive effects in the context of membrane protein manipulation.     

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction,Overstabilization, and Crystallization of Membrane Proteins

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl‐substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane–cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b , 8 c , and 9 b preserved the ATPase function of BmrA, an ATP‐binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a , 8 b , 8 f , 9 a , and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G‐protein‐coupled adenosine receptor A_2AR. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.     

Self‐Assembly Behaviors of a Penta‐Phenylene Maltoside and Its Application for Membrane Protein Study

We prepared an amphiphile with a penta‐phenylene lipophilic group and a branched trimaltoside head group. This new agent, designated penta‐phenylene maltoside (PPM), showed a marked tendency to self‐assembly into micelles via strong aromatic–aromatic interactions in aqueous media, as evidenced by ¹H NMR spectroscopy and fluorescence studies. When utilized for membrane protein studies, this new agent was superior to DDM, a gold standard conventional detergent, in stabilizing multiple proteins long term. The ability of this agent to form aromatic–aromatic interactions is likely responsible for enhanced protein stabilization when associated with a target membrane protein.     

Transition-Linker Containing Detergents for Membrane Protein Studies

It is a pressing need, but still challenging to explore the structure and function of membrane proteins (MPs). One of the main obstacles is the limited availability of matched detergents for the handling of specific MPs. We describe herein the design of new detergents by incorporation of a transition linker between the hydrophilic head and the hydrophobic tail. This design allows a gradual change of hydrophobicity between the outside and inside of micelles, in contrast to the abrupt switch in conventional detergents. Notably, many of these detergents assembled into micelles in while retaining low critical micelle concentrations. Meanwhile, thermal stabilizing evaluation identified superior detergents for representative MPs, including G protein-coupled receptors and a transporter protein. Among them, further improved the NMR study of MPs. We anticipate these that results will encourage future detergent expansion through new remodeling on the traditional detergent scaffold.     

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.     

Dendritic Oligoglycerol Regioisomer Mixtures and Their Utility for Membrane Protein Research

Dendrons are an important class of macromolecules that can be used for a broad range of applications. Recent studies have indicated that mixtures of oligoglycerol detergent (OGD) regioisomers are superior to individual regioisomers for protein extraction. The origin of this phenomenon remains puzzling. Here we discuss the synthesis and characterization of dendritic oligoglycerol regioisomer mixtures and their implementation into detergents. We provide experimental benchmarks to support quality control after synthesis and investigate the unusual utility of OGD regioisomer mixtures for extracting large protein quantities from biological membranes. We anticipate that our findings will enable the development of mixed detergent platforms in the future.     

Disulfide-Containing Detergents (DCDs) for the Structural Biology of Membrane Proteins

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A_2AAR, which is an α-helical protein, and the β-barrel protein OmpX from E. coli.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs

We present in‐membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G‐protein‐coupled receptors (GPCRs) with minimal mutagenesis. This method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site‐specific introduction of chromophores for NMR and fluorescence spectroscopy in the human κ‐opioid receptor (KOR) and the human A_2A adenosine receptor (A_2AAR). IMCM is applicable to a wide range of in vitro studies of GPCRs, including single‐molecule spectroscopy, and is a promising platform for in‐cell spectroscopy experiments.     

Outer‐Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near‐outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL^?1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37_FRRV) previously optimized as a substrate for OmpT, an outer‐membrane protease on E. coli. LL37_FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA‐LL37_FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37_FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37_FRRV sequence variants.     

Peptide‐Guided Assembly of Repeat Protein Fragments

Herein, we present the peptide‐guided assembly of complementary fragments of designed armadillo repeat proteins (dArmRPs) to create proteins that bind peptides not only with high affinity but also with good selectivity. We recently demonstrated that complementary N‐ and C‐terminal fragments of dArmRPs form high‐affinity complexes that resemble the structure of the full‐length protein, and that these complexes bind their target peptides. We now demonstrate that dArmRPs can be split such that the fragments assemble only in the presence of a templating peptide, and that fragment mixtures enrich the combination with the highest affinity for this peptide. The enriched fragment combination discriminates single amino acid variations in the target peptide with high specificity. Our results suggest novel opportunities for the generation of new peptide binders by selection from dArmRP fragment mixtures.     

Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid‐conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base‐pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE‐mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.