Travel light: Essential packing for membrane proteins with an active lifestyle.

We review the considerable progress during the recent decade in the endeavours of designing, optimising, and utilising carrier particle systems for structural and functional studies of membrane proteins in near-native environments. New and improved systems are constantly emerging, novel studies push the perceived limits of a given carrier system, and specific carrier systems consolidate and entrench themselves as the system of choice for particular classes of target membrane protein systems. This review covers the most frequently used carrier systems for such studies and emphasises similarities and differences between these systems as well as current trends and future directions for the field. Particular interest is devoted to the biophysical properties and membrane mimicking ability of each system and the manner in which this may impact an embedded membrane protein and an eventual structural or functional study.

Dark peptide discs for the investigation of membrane proteins in supported lipid bilayers: the case of synaptobrevin 2 (VAMP2).

Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both α-helical and ß-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with α-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

Structural model of tissue factor (TF) and TF-factor VIIa complex in a lipid membrane: A combined experimental and computational study.

Tissue factor (TF) is a membrane protein involved in blood coagulation. TF initiates a cascade of proteolytic reactions, ultimately leading to the formation of a blood clot. The first reaction consists of the binding of the coagulation factor VII and its conversion to the activated form, FVIIa. Here, we combined experimental, i.e. quartz crystal microbalance with dissipation monitoring and neutron reflectometry, and computational, i.e. molecular dynamics (MD) simulation, methods to derive a complete structural model of TF and TF/FVIIa complex in a lipid bilayer. This model shows that the TF transmembrane domain (TMD), and the flexible linker connecting the TMD to the extracellular domain (ECD), define the location of the ECD on the membrane surface. The average orientation of the ECD relative to the bilayer surface is slightly tilted towards the lipid headgroups, a conformation that we suggest is promoted by phosphatidylserine lipids, and favours the binding of FVIIa. On the other hand, the formation of the TF/FVIIa complex induces minor changes in the TF structure, and reduces the conformational freedom of both TF and FVIIA. Altogether we describe the protein-protein and protein-lipid interactions favouring blood coagulation, but also instrumental to the development of new drugs.

Global fitting of multiple data frames from SEC-SAXS to investigate the structure of next-generation nanodiscs.

The combination of online size-exclusion chromatography and small-angle X-ray scattering (SEC-SAXS) is rapidly becoming a key technique for structural investigations of elaborate biophysical samples in solution. Here, a novel model-refinement strategy centred around the technique is outlined and its utility is demonstrated by analysing data series from several SEC-SAXS experiments on phospholipid bilayer nanodiscs. Using this method, a single model was globally refined against many frames from the same data series, thereby capturing the frame-to-frame tendencies of the irradiated sample. These are compared with models refined in the traditional manner, in which refinement is based on the average profile of a set of consecutive frames from the same data series without an in-depth comparison of individual frames. This is considered to be an attractive model-refinement scheme as it considerably lowers the total number of parameters refined from the data series, produces tendencies that are automatically consistent between frames, and utilizes a considerably larger portion of the recorded data than is often performed in such experiments. Additionally, a method is outlined for correcting a measured UV absorption signal by accounting for potential peak broadening by the experimental setup.

Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Quantification of Conformational Entropy Unravels Effect of Disordered Flanking Region in Coupled Folding and Binding.

Intrinsic disorder (ID) constitutes a new dimension to the protein structure-function relationship. The ability to undergo conformational changes upon binding is a key property of intrinsically disordered proteins and remains challenging to study using conventional methods. A 1994 paper by R. S. Spolar and M. T. Record presented a thermodynamic approach for estimating changes in conformational entropy based on heat capacity changes, allowing quantification of residues folding upon binding. Here, we adapt the method for studies of intrinsically disordered proteins. We integrate additional data to provide a broader experimental foundation for the underlying relations and, based on >500 protein-protein complexes involving disordered proteins, reassess a key relation between polar and nonpolar surface area changes, previously determined using globular protein folding. We demonstrate the improved suitability of the adapted method to studies of the folded αα-hub domain RST from radical-induced cell death 1, whose interactome is characterized by ID. From extensive thermodynamic data, quantifying the conformational entropy changes upon binding, and comparison to the NMR structure, the adapted method improves accuracy for ID-based studies. Furthermore, we apply the method, in conjunction with NMR, to reveal hitherto undetected effects of interaction-motif context. Thus, inclusion of the disordered context of the DREB2A RST-binding motif induces structuring of the binding motif, resulting in major enthalpy-entropy compensation in the interaction interface. This study, also evaluating additional interactions, demonstrates the strength of the ID-adapted Spolar-Record thermodynamic approach for dissection of structural features of ID-based interactions, easily overlooked in traditional studies, and for translation of these into mechanistic knowledge.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Peptide discs as precursors of biologically relevant supported lipid bilayers.

Supported lipid bilayers (SLBs) are commonly used to investigate the structure and dynamics of biological membranes. Vesicle fusion is a widely exploited method to produce SLBs. However, this process becomes less favoured when the vesicles contain complex lipid mixtures, e.g. natural lipid extracts. In these cases, it is often necessary to change experimental parameters, such as temperature, to unphysiological values to trigger the SLB formation. This may induce lipid degradation and is also not compatible with including membrane proteins or other biomolecules into the bilayers. Here, we show that the peptide discs, ~10 nm discoidal lipid bilayers stabilized in solution by a self-assembled 18A peptide belt, can be used as precursors for SLBs. The characterizations by means of neutron reflectometry and attenuated total reflectance-FTIR spectroscopy show that SLBs were successfully formed both from synthetic lipid mixtures (surface coverage 90-95%) and from natural lipid mixtures (surface coverage ~85%). Traces of 18A peptide (below 0.02 M ratio) left at the support surface after the bilayer formation do not affect the SLB structure. Altogether, we demonstrate that peptide disc formation of SLBs is much faster than the SLB formation by vesicle fusion and without the need of altering any experimental variable from physiologically relevant values.

Efficient refolding and reconstitution of tissue factor into nanodiscs facilitates structural investigation of a multicomponent system on a lipid bilayer.

Structural data on membrane proteins in a lipid membrane environment is challenging to obtain but needed to provide information on the, often essential, protein-lipid interplay. A common experimental bottleneck in obtaining such data is providing samples in sufficient amounts and quality required for structural studies. We developed a new production protocol for the single-pass transmembrane protein (SPTMP) tissue factor (TF), exploiting the high expression level in E. coli inclusion bodies and subsequent refolding. This provided more than 5 mg of functional TF per liter bacterial culture. This is substantially more than what was obtained by the classical approaches for expressing TF in the membrane-anchored configuration. We optimized reconstitution into circularized nanodiscs enabling the formation of stable, TF loaded nanodiscs with different lipid compositions and with a limited material waste. The blood coagulation cascade is initiated by the complex formation between TF and Factor VIIa (FVIIa), and we probed this interaction by a functional assay and SPR measurements, which revealed similar activity and binding kinetics as TF produced by other protocols, demonstrating that high-yield production does not compromise TF function. Furthermore, the amounts of sample produced permitted initial small angle X-ray scattering studies providing the first structural information about TF and its binding to FVIIa in a lipid environment. This strategy possibly allows for probing the multicomponent complex TF:FVIIa together with its substrate Factor X on a lipid bilayer, but may also be relevant as a production strategy for other SPTMP for which structural information, in general, is limited.

Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with ∼0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

Circularized and solubility-enhanced MSPs facilitate simple and high-yield production of stable nanodiscs for studies of membrane proteins in solution.

Recently, an enzymatic reaction was utilized to covalently link the N and C termini of membrane scaffold proteins to produce circularized nanodiscs that were more homogeneous and stable than standard nanodiscs. We continue this development and aim for obtaining high yields of stable and monodisperse nanodiscs for structural studies of membrane proteins by solution small-angle scattering techniques. Based on the template MSP1E3D1, we designed an optimized membrane scaffold protein (His-lsMSP1E3D1) with a sortase recognition motif and high abundance of solubility-enhancing negative charges. With these modifications, we show that high protein expression is maintained and that the circularization reaction is efficient, such that we obtain a high yield of circularized membrane scaffold protein (csMSP1E3D1) and downstream circularized nanodiscs. We characterize the circularized protein and corresponding nanodiscs biophysically by small-angle X-ray scattering, size-exclusion chromatography, circular dichroism spectroscopy, and light scattering and compare to noncircularized samples. First, we show that circularized and noncircularized (lsMSP1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that lsMSP1E3D1 nanodiscs are more stable compared to the template MSP1E3D1 nanodiscs as an effect of the extra negative charges and that csMSP1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in csMSP1E3D1 nanodiscs. Large-scale production methods for circularized nanodiscs with improved thermal and temporal stability will facilitate better access to the nanodisc technology and enable applications at physiologically relevant temperatures.

Structures and Short Linear Motif of Disordered Transcription Factor Regions Provide Clues to the Interactome of the Cellular Hub Protein Radical-induced Cell Death1.

Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein radical-induced cell death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound by RCD1, we analyzed the IDRs in three protein partners, DREB2A (dehydration-responsive element-binding protein 2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as (DE)X(1,2)(YF)X(1,4)(DE)L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher Kd values. Specifically, although the SLiM was surrounded by IDRs, individual intrinsic α-helix propensities varied as shown by CD spectroscopy. NMR spectroscopy further demonstrated that DREB2A underwent coupled folding and binding with α-helix formation upon interaction with RCD1, whereas peptides from ANAC013 and ANAC046 formed different structures or were fuzzy in the complexes. These findings allow us to present a model of the stress-associated RCD1-transcription factor interactome and to contribute to the emerging understanding of the interactions between folded hubs and their intrinsically disordered partners.