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.

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.

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.

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.

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs.

Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nanodiscs.

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.

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge.

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios.

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.

Influence of different polymer belts on lipid properties in nanodiscs characterized by CW EPR spectroscopy.

With this study we aim at comparing the well-known lipid membrane model system of liposomes and polymer-encapsulated nanodiscs regarding their lipid properties. Using differential scanning calorimetry (DSC) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we characterize the temperature-dependent lipid behavior within 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes and nanodiscs made from such liposomes by application of various polymers based on styrene-co-maleic acid (SMA), diisobutylene-alt-maleic acid (DIBMA), and styrene-co-maleic amide sulfobetaine (SMA-SB), a new SMA-derived copolymer containing sulfobetaine side chains. By incorporating a spin label doxyl moiety into the lipid bilayer in position 16 or 5 we were able to study the micropolarity as well as rotational restrictions onto the lipids in the apolar bilayer center and the chain region adjacent to the carbonyl groups, respectively. Our results suggest that all polymers broaden the main melting transition of DMPC, change the water accessibility within the lipid bilayer, and exhibit additional constraints onto the lipids. Independent of the used polymer, the rotational mobility of both spin-labeled lipids decreased with DIBMA exerting less restraints probably due to its aliphatic side chains. Our findings imply that the choice of the solubilizing polymer has to be considered an important step to form lipid nanodiscs which should be included into research of lipid membranes and membrane proteins in the future.

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.

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.

Hydrogenated Diglucose Detergents for Membrane-Protein Extraction and Stabilization.

We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane-, and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents, respectively. Micellization in aqueous solution was studied by isothermal titration calorimetry, 1H NMR spectroscopy, and surface tensiometry. Critical micellar concentration values were found to decrease by a factor of ∼10 for each pair of methylene groups added to the alkyl chain, ranging from ∼0.05 to 9 mM for DDDG and ODG, respectively. Dynamic light scattering and analytical ultracentrifugation sedimentation velocity experiments were used to investigate the size and composition of the micellar aggregates, showing that the aggregation number significantly increased from ∼40 for ODG to ∼80 for DDDG. All new compounds were able to solubilize membrane proteins (MPs) from bacterial membranes, insect cells, as well as the Madin-Darby canine kidney cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter (BmrA) were solubilized efficiently. Striking thermostability improvements of +13 and +8 °C were observed when ODG and DDG were, respectively, applied to wild-type and full-length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of membrane proteins (MPs) and thus makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging MP targets.

Fluorinated diglucose detergents for membrane-protein extraction.

Fluorinated surfactants have scarcely been explored for the direct extraction of proteins from membranes because fluorination is believed to abrogate detergency. However, we have recently shown that a commercially available fluorinated surfactant readily solubilizes lipid membranes, thereby suggesting that fluorination per se does not interfere with detergent activity. In this work, we developed new fluorinated surfactants that exhibit detergency in terms of both lipid-vesicle solubilization and membrane-protein extraction. The compounds made and tested contain two glucose moieties as polar headgroup, a hydrogenated thioether linker, and a perfluorinated alkyl tail with either 4, 6, or 8 carbon atoms. The physicochemical properties of the micelles formed by the three fluorinated surfactants were evaluated by NMR spectroscopy, surface tensiometry, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. At 25 °C, micellization was mainly entropy-driven, and the CMC values were found to decrease with chain length of the fluorinated tail, whereas the aggregation number increased with chain length. Remarkably, all three surfactants were found to solubilize lipid vesicles and extract a broad range of proteins from Escherichia coli membranes. These findings demonstrate, for the first time, that nonionic fluorinated surfactants could be further exploited for the direct extraction and solubilization of membrane proteins.

Extracavity Effect in Cyclodextrin/Surfactant Complexation.

Cyclodextrin (CD) complexation is a convenient method to sequester surfactants in a controllable way, for example, during membrane-protein reconstitution. Interestingly, the equilibrium stability of CD/surfactant inclusion complexes increases with the length of the nonpolar surfactant chain even beyond the point where all hydrophobic contacts within the canonical CD cavity are saturated. To rationalize this observation, we have dissected the inclusion complexation equilibria of a structurally well-defined CD, that is, heptakis(2,6-di- O-methyl)-ß-CD (DIMEB), and a homologous series of surfactants, namely, n-alkyl- N, N-dimethyl-3-ammonio-1-propanesulfonates (SB3- x) with chain lengths ranging from x = 8 to 14. Thermodynamic parameters obtained by isothermal titration calorimetry and structural insights derived from nuclear magnetic resonance spectroscopy and molecular dynamics simulations revealed that, upon inclusion, long-chain surfactants with x = ≥10 extend beyond the canonical CD cavity. This enables the formation of hydrophobic contacts between long surfactant chains and the extracavity parts of DIMEB, which make additional favorable contributions to the stability of the inclusion complex. These results explain the finding that the stability of CD/surfactant inclusion complexes monotonously increases with the surfactant chain length even for long chains that completely fill the canonical CD cavity.

Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer.

Membrane proteins usually need to be extracted from their native environment and separated from other membrane components for in-depth in vitro characterization. The use of styrene/maleic acid (SMA) copolymers to solubilize membrane proteins and their surrounding lipids into bilayer nanodiscs is an attractive approach toward this goal. We have recently shown that a diisobutylene/maleic acid (DIBMA) copolymer similarly solubilizes model and cellular membranes but, unlike SMA(3:1), has a mild impact on lipid acyl-chain order and thermotropic phase behavior. Here, we used fluorescence spectroscopy, small-angle X-ray scattering, size-exclusion chromatography, dynamic light scattering, and 31P nuclear magnetic resonance spectroscopy to examine the self-association of DIBMA and its membrane-solubilization properties against lipids differing in acyl-chain length and saturation. Although DIBMA is less hydrophobic than commonly used SMA(3:1) and SMA(2:1) copolymers, it efficiently formed lipid-bilayer nanodiscs that decreased in size with increasing polymer/lipid ratio while maintaining the overall thickness of the membrane. DIBMA fractions of different molar masses were similarly efficient in solubilizing a saturated lipid. Coulomb screening at elevated ionic strength or reduced charge density on the polymer at low pH enhanced the solubilization efficiency of DIBMA. The free-energy penalty for transferring phospholipids from vesicular bilayers into nanodiscs became more unfavorable with increasing acyl-chain length and unsaturation. Altogether, these findings provide a rational framework for using DIBMA in membrane-protein research by shedding light on the effects of polymer and lipid properties as well as experimental conditions on membrane solubilization.

Thermodynamics of nanodisc formation mediated by styrene/maleic acid (2:1) copolymer.

Styrene/maleic acid copolymers (SMA) have recently attracted great interest for in vitro studies of membrane proteins, as they self-insert into and fragment biological membranes to form polymer-bounded nanodiscs that provide a native-like lipid-bilayer environment. SMA copolymers are available in different styrene/maleic acid ratios and chain lengths and, thus, possess different charge densities, hydrophobicities, and solubilisation properties. Here, we studied the equilibrium solubilisation properties of the most commonly used copolymer, SMA(2:1), by monitoring the formation of nanodiscs from phospholipid vesicles using 31P nuclear magnetic resonance spectroscopy, dynamic light scattering, and differential scanning calorimetry. Comparison of SMA(2:1) phase diagrams with those of SMA(3:1) and diisobutylene/maleic acid (DIBMA) revealed that, on a mass concentration scale, SMA(2:1) is the most efficient membrane solubiliser, despite its relatively mild effects on the thermotropic phase behaviour of solubilised lipids. In contrast with previous kinetic studies, our equilibrium experiments demonstrate that the solubilisation of phospholipid bilayers by SMA(2:1) is most efficient at moderately alkaline pH values. This pH dependence was also observed for the solubilisation of native Escherichia coli membranes, for which SMA(2:1) again turned out to be the most powerful solubiliser in terms of the total amounts of membrane proteins extracted.

Model-Free Analysis of Critical Micellar Concentrations for Detecting Demixing in Surfactant Mixtures.

Aqueous mixtures of two or more surfactants are often employed for research or industrial purposes because such mixtures offer advantages over single-surfactant systems. This is particularly true for mixtures of fluorocarbon (FC) and hydrocarbon (HC) surfactants, which display a broad range of mutual miscibilities in mixed micelles. Unfortunately, the prediction and even the experimental elucidation of the micellar mixing behavior of surfactant mixtures remain challenging, as evidenced by conflicting results and conclusions derived from diverse, and often complex, mixing models. One of the most intriguing questions is whether certain combinations of FC and HC surfactants form only one type of mixed micelle or rather demix into two micelle populations, namely, FC-rich and HC-rich ones. Here, we demonstrate a novel approach to the model-free analysis of critical micellar concentrations (CMCs) of surfactant mixtures that is based on a fit of the experimental data with cubic splines using a stringent thermodynamic criterion for mixing. As a proof of principle, we analyze CMC values determined by isothermal titration calorimetry and confirm the conclusions with the aid of combined 1H- and 19F-NMR spectroscopy. Specifically, we show that aqueous mixtures of an FC maltoside and an HC maltoside conform with the assumption of only one type of micelle regardless of the mixing ratio, whereas combining the same FC surfactant with an HC surfactant carrying a zwitterionic phosphocholine headgroup gives rise to two coexisting micelle populations at high mole fractions of the FC maltoside.

Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.

Influence of lipid bilayer properties on nanodisc formation mediated by styrene/maleic acid copolymers.

Copolymers of styrene and maleic acid (SMA) have gained great attention as alternatives to conventional detergents, as they offer decisive advantages for studying membrane proteins and lipids in vitro. These polymers self-insert into artificial and biological membranes and, at sufficiently high concentrations, solubilise them into disc-shaped nanostructures containing a lipid bilayer core surrounded by a polymer belt. We have used (31)P nuclear magnetic resonance spectroscopy and dynamic light scattering to systematically study the solubilisation of vesicles composed of saturated or unsaturated phospholipids by an SMA copolymer with a 3 : 1 styrene/maleic acid molar ratio at different temperatures. Solubilisation was thermodynamically rationalised in terms of a three-stage model that treats various lipid/polymer aggregates as pseudophases. The solubilising capacity of SMA(3 : 1) towards a saturated lipid is higher in the gel than in the liquid-crystalline state of the membrane even though solubilisation is slower. Although the solubilisation of mixed fluid membranes is non-selective, the presence of a non-bilayer phospholipid lowers the threshold at which the membrane becomes saturated with SMA(3 : 1) but raises the polymer concentration required for complete solubilisation. Both of these trends can be explained by considering the vesicle-to-nanodisc transfer free energies of the lipid and the polymer. On the basis of the phase diagrams thus obtained, re-association of polymer-solubilised lipids with vesicles is possible under mild conditions, which has implications for the reconstitution of proteins and lipids from nanodiscs into vesicular membranes. Finally, the phase diagrams provide evidence for the absence of free SMA(3 : 1) in vesicular lipid suspensions.

Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions.

Isothermal titration calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.