Exploring membrane asymmetry and its effects on membrane proteins.

Plasma membranes utilize free energy to maintain highly asymmetric, non-equilibrium distributions of lipids and proteins between their two leaflets. In this review we discuss recent progress in quantitative research enabled by using compositionally controlled asymmetric model membranes. Both experimental and computational studies have shed light on the nuanced mechanisms that govern the structural and dynamic coupling between compositionally distinct bilayer leaflets. This coupling can increase the membrane bending rigidity and induce order - or lipid domains - across the membrane. Furthermore, emerging evidence indicates that integral membrane proteins not only respond to asymmetric lipid distributions but also exhibit intriguing asymmetric properties themselves. We propose strategies to advance experimental research, aiming for a deeper, quantitative understanding of membrane asymmetry, which carries profound implications for cellular physiology.

Botrytis hypersensitive response inducing protein 1 triggers noncanonical PTI to induce plant cell death.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

Dimerization and Crowding in the Binding of Interleukin 8 to Dendritic Glycosaminoglycans as Artificial Proteoglycans.

The interactions of glycosaminoglycans (GAG) with proteins of the extracellular matrix govern and regulate complex physiological functions including cellular growth, immune response, and inflammation. Repetitive presentation of GAG binding motifs, as found in native proteoglycans, might enhance GAG-protein binding through multivalent interactions. Here, we report the chemical synthesis of dendritic GAG oligomers constructed of nonasulfated hyaluronan tetrasaccharides for investigating the binding of the protein chemokine interleukin 8 (IL-8) to artificial, well-defined proteoglycan architectures. Binding of mutant monomeric and native dimerizable IL-8 was investigated by NMR spectroscopy and isothermal titration calorimetry. Dendritic oligomerization of GAG increased the binding affinity of both monomeric and dimeric IL-8. Monomeric IL-8 bound to monomeric and dimeric GAG with KD values of 7.3 and 0.108 µM, respectively. The effect was less pronounced for dimerizable wild-type IL-8, for which GAG dimerization improved the affinity from 34 to 5 nM. Binding of dimeric IL-8 to oligomeric GAG was limited by steric crowding effects, strongly reducing the affinity of subsequent binding events. In conclusion, the strongest effect of GAG oligomerization was the amplified binding of IL-8 monomers, which might concentrate monomeric protein in the extracellular matrix and thus promote protein dimerization under physiological conditions.

Towards a Light-mediated Gene Therapy for the Eye using Caged Ethinylestradiol and the Inducible Cre/lox System.

Increasingly, retinal pathologies are being treated with virus-mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e., "caging." Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP-CreERT2 mice with a Cre-dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light-mediated gene therapy for the eyes.

FULL-MDS: Fluorescent Universal Lipid Labeling for Microfluidic Diffusional Sizing.

Microfluidic diffusional sizing (MDS) is a recent and powerful method for determining the hydrodynamic sizes and interactions of biomolecules and nanoparticles. A major benefit of MDS is that it can report the size of a fluorescently labeled target even in mixtures with complex, unpurified samples. However, a limitation of MDS is that the target itself has to be purified and covalently labeled with a fluorescent dye. Such covalent labeling is not suitable for crude extracts such as native nanodiscs directly obtained from cellular membranes. In this study, we introduce fluorescent universal lipid labeling for MDS (FULL-MDS) as a sparse, noncovalent labeling method for determining particle size. We first demonstrate that the inexpensive and well-characterized fluorophore, Nile blue, spontaneously partitions into lipid nanoparticles without disrupting their structure. We then highlight the key advantage of FULL-MDS by showing that it yields robust size information on lipid nanoparticles in crude cell extracts that are not amenable to other sizing methods. Furthermore, even for synthetic nanodiscs, FULL-MDS is faster, cheaper, and simpler than existing labeling schemes.

Lipid exchange among electroneutral Sulfo-DIBMA nanodiscs is independent of ion concentration.

Polymer-encapsulated nanodiscs enable membrane proteins to be investigated within a native-like lipid-bilayer environment. Unlike other bilayer-based membrane mimetics, these nanodiscs are equilibrium structures that permit lipid exchange on experimentally relevant timescales. Therefore, examining the kinetics and mechanisms of lipid exchange is of great interest. Since the high charge densities of existing anionic polymers can interfere with protein-protein and protein-lipid interactions as well as charge-sensitive analysis techniques, electroneutral nanodisc-forming polymers have been recently introduced. However, it has remained unclear how the electroneutrality of these polymers affects the lipid-exchange behavior of the nanodiscs. Here, we use time-resolved Förster resonance energy transfer to study the kinetics and the mechanisms of lipid exchange among nanodiscs formed by the electroneutral polymer Sulfo-DIBMA. We also examine the role of coulombic repulsion and specific counterion association in lipid exchange. Our results show that Sulfo-DIBMA nanodiscs exchange lipids on a similar timescale as DIBMA nanodiscs. In contrast with nanodiscs made from polyanionic DIBMA, however, the presence of mono- and divalent cations does not influence lipid exchange among Sulfo-DIBMA nanodiscs, as expected from their electroneutrality. The robustness of Sulfo-DIBMA nanodiscs against varying ion concentrations opens new possibilities for investigating charge-sensitive processes involving membrane proteins.

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles.

Different amphiphilic co-polymers have been introduced to produce polymer-lipid particles with nanodisc structure composed of an inner lipid bilayer and polymer chains self-assembled as an outer belt. These particles can be used to stabilize membrane proteins in solution and enable their characterization by means of biophysical methods, including small-angle X-ray scattering (SAXS). Some of these co-polymers have also been used to directly extract membrane proteins together with their associated lipids from native membranes. Styrene/maleic acid and diisobutylene/maleic acid are among the most commonly used co-polymers for producing polymer-lipid particles, named SMALPs and DIBMALPs, respectively. Recently, a new co-polymer, named Glyco-DIBMA, was produced by partial amidation of DIBMA with the amino sugar N-methyl-d-glucosamine. Polymer-lipid particles produced with Glyco-DIBMA, named Glyco-DIBMALPs, exhibit improved structural properties and stability compared to those of SMALPs and DIBMALPs while retaining the capability of directly extracting membrane proteins from native membranes. Here, we characterize the structure and lipid composition of Glyco-DIBMALPs produced with either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Glyco-DIBMALPs were also prepared with mixtures of either POPC or DMPC and cholesterol at different mole fractions. We estimated the lipid content in the Glyco-DIBMALPs and determined the particle structure and morphology by SAXS. We show that the Glyco-DIBMALPs are nanodisc-like particles whose size and shape depend on the polymer/lipid ratio. This is relevant for designing nanodisc particles with a tunable diameter according to the size of the membrane protein to be incorporated. We also report that the addition of >20 mol % cholesterol strongly perturbed the formation of Glyco-DIBMALPs. Altogether, we describe a detailed characterization of the Glyco-DIBMALPs, which provides relevant inputs for future application of these particles in the biophysical investigation of membrane proteins.

Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment.

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.

Self-Assembly of Protein-Containing Lipid-Bilayer Nanodiscs from Small-Molecule Amphiphiles.

When membrane proteins are removed from their natural environment, the quality of the membrane-solubilizing agent used is critical for preserving their native structures and functions. Nanodiscs that retain a lipid-bilayer core around membrane proteins have attracted great attention because they offer a much more native-like environment than detergent micelles. Here, two small-molecule amphiphiles with diglucose headgroups and either a hydrocarbon or a fluorocarbon hydrophobic chain are shown to directly assemble lipids and membrane proteins to form native nanodiscs rather than mixed micelles. Self-assembly of nanodiscs of increasing complexity from both defined, artificial vesicles as well as complex, cellular membranes is demonstrated. A detailed investigation of bilayer integrity and membrane-protein activity in these nanodiscs reveals gentle effects on the encapsulated bilayer core. The fluorinated amphiphile appears particularly promising because its lipophobicity results in gentle, non-perturbing interactions with the nanoscale lipid bilayer. A sequential model of nanodisc self-assembly is proposed that proceeds through perforation of the original membrane followed by saturation and complete solubilization of the bilayer. On this basis, pseudophase diagrams are established for mixtures of lipids and nanodisc-forming diglucoside amphiphiles, and the latter are used for the extraction of a broad range of membrane proteins from cellular membranes.

Maltose-Based Fluorinated Surfactants for Membrane-Protein Extraction and Stabilization.

Two new surfactants, F5OM and F5DM, were designed as partially fluorinated analogues of n-dodecyl-ß-D-maltoside (DDM). The micellization properties and the morphologies of the aggregates formed by the two surfactants in water and phosphate buffer were evaluated by NMR spectroscopy, surface tension measurement, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. As expected, the critical micellar concentration (cmc) was found to decrease with chain length of the fluorinated tail from 2.1-2.5 mM for F5OM to 0.3-0.5 mM for F5DM, and micellization was mainly entropy-driven at 25 °C. Close to their respective cmc, the micelle sizes were similar for both surfactants, that is, 7 and 13 nm for F5OM and F5DM, respectively, and both increased with concentration forming 4 nm diameter rods with maximum dimensions of 50 and 70 nm, respectively, at a surfactant concentration of ∼30 mM. The surfactants were found to readily solubilize lipid vesicles and extract membrane proteins directly from Escherichia coli membranes. They were found more efficient than the commercial fluorinated detergent F6H2OM over a broad range of concentrations (1-10 mM) and even better than DDM at low concentrations (1-5 mM). When transferred into the two new surfactants, the thermal stability of the proteins bacteriorhodopsin (bR) and FhuA was higher than in the presence of their solubilization detergents and similar to that in DDM; furthermore, bR was stable over several months. The membrane enzymes SpNOX and BmrA were not as active as in DDM micelles but similarly active as in F6OM. Together, these findings indicate both extracting and stabilizing properties of the new maltose-based fluorinated surfactants, making them promising tools in MP applications.

Nanoscale Model System for the Human Myelin Sheath.

Neurodegenerative disorders are among the most common diseases in modern society. However, the molecular bases of diseases such as multiple sclerosis or Charcot-Marie-Tooth disease remain far from being fully understood. Research in this field is limited by the complex nature of native myelin and by difficulties in obtaining good in vitro model systems of myelin. Here, we introduce an easy-to-use model system of the myelin sheath that can be used to study myelin proteins in a native-like yet well-controlled environment. To this end, we present myelin-mimicking nanodiscs prepared through one of the amphiphilic copolymers styrene/maleic acid (SMA), diisobutylene/maleic acid (DIBMA), and styrene/maleimide sulfobetaine (SMA-SB). These nanodiscs were tested for their lipid composition using chromatographic (HPLC) and mass spectrometric (MS) methods and, utilizing spin probes within the nanodisc, their comparability with liposomes was studied. In addition, their binding behavior with bovine myelin basic protein (MBP) was scrutinized to ensure that the nanodiscs represent a suitable model system of myelin. Our results suggest that both SMA and SMA-SB are able to solubilize the myelin-like (cytoplasmic) liposomes without preferences for specific lipid headgroups or fatty acyl chains. In nanodiscs of both SMA and SMA-SB (called SMA(-SB)-lipid particles, short SMALPs or SMA-SBLPs, respectively), the polymers restrict the lipids' motion in the hydrophobic center of the bilayer. The headgroups of the lipids, however, are sterically less hindered in nanodiscs when compared with liposomes. Myelin-like SMALPs are able to bind bovine MBP, which can stack the lipid bilayers like in native myelin, showing the usability of these simple, well-controlled systems in further studies of protein-lipid interactions of native myelin.

A multi-laboratory benchmark study of isothermal titration calorimetry (ITC) using Ca2+ and Mg2+ binding to EDTA.

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

Hybrid Fluorocarbon-Hydrocarbon Surfactants: Synthesis and Colloidal Characterization.

Four double-tailed hybrid fluorocarbon-hydrocarbon (F-H) surfactants with a poly(ethylene glycol) (PEG) polar headgroup were synthesized. The hydrophobic scaffold consists of an amino acid core, onto which were grafted both fluorocarbon and hydrocarbon chains of different lengths. The PEG polar head was connected to the hydrophobic scaffold through a copper(I)-mediated click reaction. The four derivatives exhibit aqueous solubility >100 g/L and self-assemble into micellar aggregates with micromolar critical micellar concentration (CMC) values, as demonstrated by isothermal titration calorimetry (ITC), surface tension (ST) measurements, and steady-state fluorescence spectroscopy. The CMC value decreased by a factor of ∼6 for each additional pair of CH2 groups, whereas a decrease by a factor of ∼2.5 was observed when the size of the PEG polar head was reduced from 2000 to 750 g/mol. Dynamic light scattering (DLS) showed unimodal micelle populations with hydrodynamic diameters of 10-15 nm, in agreement with results obtained from size-exclusion chromatography (SEC). The aggregation number increased with the hydrocarbon chain length but decreased with increasing PEG chain lengths. The combination in one molecular design of both low CMC and high water solubility makes these new surfactants promising systems for novel drug-delivery systems.

Lipid exchange among polymer-encapsulated nanodiscs by time-resolved Förster resonance energy transfer.

Some amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) copolymers are able to directly extract cellular membranes into nanosized polymer-bounded lipid-bilayer patches. These polymer-encapsulated nanodiscs offer the possibility to investigate delicate membrane proteins along with their surrounding lipids and, thus, protein/lipid interactions, in a near-native bilayer environment. By dissecting the kinetics of lipid exchange among DIBMA- and SMA-bounded nanodiscs, we have recently shown that the encapsulated lipid bilayer does not represent a static snapshot of the membrane at the time point of solubilisation. Instead, nanoscale lipid-bilayer patches remain in equilibrium with one another through lipid exchange enabled by nanodisc collisions. This finding is important for correctly interpreting any attempts at studying protein/lipid interactions with the aid of polymer-based nanodiscs and will be relevant to characterising the rapidly growing repertoire of new amphiphilic copolymers for membrane extraction. A highly sensitive and efficient technique for measuring the kinetics of lipid transfer among various kinds of nanosized membrane mimics consists in time-resolved Förster resonance energy transfer (TR-FRET) monitored in a stopped-flow apparatus. Here, we provide detailed instructions on how to measure the kinetics and unravel the underlying mechanisms of lipid exchange among lipid-bilayer nanodiscs under various solution conditions.

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.

Fast and Robust Quantification of Detergent Micellization Thermodynamics from Isothermal Titration Calorimetry.

Detergents are widely used in modern in vitro biochemistry and biophysics, in particular to aid the characterization of integral membrane proteins. An important characteristic of these chemicals in aqueous solutions is the concentration above which their molecular monomers self-associate to form micelles, termed the critical micellar concentration (CMC). Micelles are supramolecular assemblies arranged with the hydrophobic portions oriented inward and the hydrophilic head groups positioned outward to interact with the aqueous solvent. Knowledge of the CMC is not only of practical relevance but also of theoretical interest because it provides thermodynamic insights. Isothermal titration calorimetry (ITC) is a powerful method to determine CMCs, as it furnishes additional information on the enthalpy and entropy of micellization. Here we describe our extension of previous methods to determine CMCs and other thermodynamic parameters from ITC demicellization curves. The new algorithm, incorporated into the stand-alone software package D/STAIN, analyzes ITC demicellization curves by taking advantage of state-of-the-art thermogram-integration techniques and automatically providing rigorous confidence intervals on the refined parameters. As a demonstration of the software's capabilities, we undertook ITC experiments to determine the respective CMCs of n-octyl ß-d-glucopyranoside (OG), n-dodecyl ß-d-maltopyranoside (DDM), and lauryldimethylamine N-oxide (LDAO). Motivated by the fact that in vitro membrane protein studies often require additives such as precipitants (e.g., polyethylene glycol (PEG)), we also carried out ITC demicellization studies in the presence of PEG3350, finding in all cases that PEG had significant effects on the thermodynamics of detergent micellization.

The Role of Plastidic Trigger Factor Serving Protein Biogenesis in Green Algae and Land Plants.

Biochemical processes in chloroplasts are important for virtually all life forms. Tight regulation of protein homeostasis and the coordinated assembly of protein complexes, composed of both imported and locally synthesized subunits, are vital to plastid functionality. Protein biogenesis requires the action of cotranslationally acting molecular chaperones. One such chaperone is trigger factor (TF), which is known to cotranslationally bind most newly synthesized proteins in bacteria, thereby assisting their correct folding and maturation. However, how these processes are regulated in chloroplasts remains poorly understood. We report here functional investigation of chloroplast-localized TF (TIG1) in the green alga (Chlamydomonas reinhardtii) and the vascular land plant Arabidopsis (Arabidopsis thaliana). We show that chloroplastic TIG1 evolved as a specialized chaperone. Unlike other plastidic chaperones that are functionally interchangeable with their prokaryotic counterpart, TIG1 was not able to complement the broadly acting ortholog in Escherichia coli. Whereas general chaperone properties such as the prevention of aggregates or substrate recognition seems to be conserved between bacterial and plastidic TFs, plant TIG1s differed by associating with only a relatively small population of translating ribosomes. Furthermore, a reduction of plastidic TIG1 levels leads to deregulated protein biogenesis at the expense of increased translation, thereby disrupting the chloroplast energy household. This suggests a central role of TIG1 in protein biogenesis in the chloroplast.

Copper and zinc ions govern the trans-directed dimerization of APP family members in multiple ways.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein 1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growth factor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and in vitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

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.