Volume 6, Issue 1 - Special issue: Lipid Nanotechnologies for Macromolecular Structure Determination / Guest Editor: Svetla Stoilova-McPhie

Due to their unique properties, tunable size, and ability to provide a near native lipid environment, nanodiscs have found widespread use for the structural and functional studies of reconstituted membrane proteins. They have also been developed, albeit in a few applications, for therapeutic and biomedical use. For these studies and applications, it is essential to characterize the nanodisc preparations in terms of their monodispersity, size, and composition, as these can influence the properties of the membrane protein of interest. Of the many biophysical methods utilized for the study and characterization of nanodiscs, we show that analytical ultracentrifugation is able to report on sample homogeneity, shape, size, composition, and membrane protein stoichiometry or oligomerization state in a direct and simple fashion. The method is truly versatile and does not require nanodisc modification or disassembly.

Membrane proteins are one of the most challenging and attractive objects in modern structural biology, as they are targets for the majority of medicines. However, studies of membrane proteins are hindered by several obstacles, including their low ability to crystallize, highly dynamic behavior of some of their domains, and need for membrane-like environment. Although solution nuclear magnetic resonance (NMR) is a very powerful technique of structural biology in terms of the amount of provided data, it imposes several limitations on the object under investigation, with the main constraint being related to the size of the object. For this reason, the membrane mimetic has to form particles of small size and simultaneously to properly simulate the bilayer membrane to be applicable for solution NMR spectroscopy. Here we review the recent advances in the field of membrane mimetics for solution NMR studies, discuss the advantages and drawbacks of specific membrane-like environments, and formulate the criteria for the selection of proper environment for a particular membrane protein or domain.

ATP-binding cassette (ABC) exporters transport substrates across biological membranes using ATP hydrolysis by a process that involves switching between inward- and outward-facing conformations. Most of the structural studies of ABC proteins have been performed with proteins in detergent micelles, locked in specific conformations and/or at low temperature. In this article, we present recent data from our laboratories where we studied the prototypical ABC exporter MsbA during ATP hydrolysis, at 37°C, reconstituted in a lipid bilayer. These studies were possible through the use of luminescence resonance energy transfer spectroscopy in MsbA reconstituted in nanodiscs. We found major differences between MsbA in these native-like conditions and in previous studies. These include a separation between the nucleotide-binding domains that was much smaller than previously thought, and a large fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis, the molecular mechanisms of which have been a subject of intensive study. This process is important for therapeutic intervention in various diseases, such as cancer. Pro-apoptotic Bax and Bak are functionally redundant and structurally homologous. When activated at the mitochondrial outer membrane, they cause the membrane to permeabilize and release apoptogenic proteins from the intermembrane space. To unravel the molecular mechanisms of this unique and important event, we systematically reduced the experimental system. Simple outer membrane vesicles and liposomes recapitulated many features of MOMP. Although conventional transmission electron microscopy could not detect any membrane changes during MOMP in these vesicles, cryo-electron microscopy successfully revealed Bax-induced delicate pores, owing to its ability to preserve native, hydrated membrane structure. The data are consistent with the idea that Bax is unfolded and embedded in the bilayer and deforms the membrane to form a large pore. Together with the biochemical and structure data in the literature, we now have more comprehensive models of the key function of Bax. We hope that new tools, such as lipid nanodiscs, will give us an atomic-level resolution and finally solve Bax structure in the membrane, where it functions.

Membrane proteins are vital to life and major therapeutic targets. Yet, understanding how they function is limited by a lack of structural information. In biological cells, membrane proteins reside in lipidic membranes and typically experience different buffer conditions on both sides of the membrane or even electric potentials and transmembrane gradients across the membranes. Proteoliposomes, which are lipidic vesicles filled with reconstituted membrane proteins, provide an ideal model system for structural and functional studies of membrane proteins under conditions that mimic nature to a certain degree. We discuss methods for the formation of liposomes and proteoliposomes, their imaging by cryo-electron microscopy, and the structural analysis of proteins present in their bilayer. We suggest the formation of ordered arrays akin to weakly ordered two-dimensional (2D) crystals in the bilayer of liposomes as a means to achieve high-resolution, and subsequent buffer modification as a method to capture snapshots of membrane proteins in action.

Cellular membranes and associated proteins play critical physiological roles in organisms from all life kingdoms. In many cases, malfunction of biological membranes triggered by changes in the lipid bilayer properties or membrane protein functional abnormalities lead to severe diseases. To understand in detail the processes that govern the life of cells and to control diseases, one of the major tasks in biological sciences is to learn how the membrane proteins function. To do so, a variety of biochemical and biophysical approaches have been used in molecular studies of membrane protein structure and function on the nanoscale. This review focuses on electron paramagnetic resonance with site-directed nitroxide spin-labeling (SDSL EPR), which is a rapidly expanding and powerful technique reporting on the local protein/spin-label dynamics and on large functionally important structural rearrangements. On the other hand, adequate to nanoscale study membrane mimetics have been developed and used in conjunction with SDSL EPR. Primarily, these mimetics include various liposomes, bicelles, and nanodiscs. This review provides a basic description of the EPR methods, continuous-wave and pulse, applied to spin-labeled proteins, and highlights several representative applications of EPR to liposome-, bicelle-, or nanodisc-reconstituted membrane proteins.

Out of all membrane mimetics available for solution nuclear magnetic resonance (NMR) spectroscopy, phospholipid bicelles are the most prospective. Unlike lipid-protein nanodiscs their size can be easily controlled over a wide range, and the exchange of matter between the particles can take place. However, recent studies revealed several major drawbacks of conventional 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and DMPC/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) bicelles. First, size of such bicelles can increase dramatically upon heating, and, second, rim-forming detergents of bicelles can cause improper folding of the water-soluble globular domains of membrane proteins. In order to avoid these effects, we tested the Façade detergents as possible alternative rim-forming agents for small isotropic bicelles. In the present work we characterized the size of bicelles formed by 3α-hydroxy-7α,12α-di-((O-β-D-maltosyl)-2-hydroxyethoxy)-cholane (Façade-EM) and 3α-hydroxy-7α,12α-di-(((2-(trimethylamino)ethyl)phosphoryl)ethyloxy)-cholane Façade-EPC as a function of temperature and lipid/detergent ratio by 1 H NMR diffusion spectroscopy. Additionally, the denaturing effects of these two rim-forming agents were investigated using the junction of the transmembrane and intracellular domains of the p75 neurotrophin receptor (p75NTR) as a model object. We show that the use of Façades allows decreasing the temperature-dependent growth of bicelles. The ability of Façade-EM-based bicelles to support the native structure and soluble state of the p75NTR intracellular domain was also revealed.

Apolipoprotein nanodiscs are a versatile tool in nanotechnology as membrane mimetics allowing, for example, the study of membrane proteins. It has recently been discovered that the Parkinson’s disease associated protein α-synuclein (α-Syn) can also form discoid-like lipoprotein nanoparticles. The present review highlights the observation that α-Syn has the properties to define stable and homogeneous populations of nanoparticles with diameters of 7–10 nm and 19–28 nm by modifying lipid vesicles or encapsulating lipid bilayers in a nanodisc-type fashion, respectively. In contrast to apolipoprotein nanodiscs, α-Syn nanoparticles can incorporate entirely negatively charged lipids emphasizing their potential use in nanotechnology as a negatively charged membrane mimetic.

Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water-soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques. Nuclear magnetic resonance is one such high-resolution approach that looks at the structure and dynamics of a protein with atomic level precision. Recently, there has been a breakthrough in making nanodiscs more susceptible for structure determination by solution NMR, yet it still remains to become the preferred choice for a membrane mimetic. In this practical review, we provide a general discourse on nanodisc and its application to solution NMR. We also offer potential solutions to remediate the technical challenges associated with nanodisc preparation and the choice of proper experimental set-ups. Along with discussing several structural applications, we demonstrate an alternative use of nanodiscs for functional studies, where we investigated the phosphorylation of a cell surface receptor, integrin. This is the first successful manifestation of observing activated receptor phosphorylation in nanodisc through NMR. We additionally present an on-column method for nanodisc preparation with multiple strategies and discuss the potential use of alternative nanoscale phospholipid bilayer systems like styrene maleic acid lipid disc and saposin-A lipoprotein disc.

Biological membranes surround all living cells, confining internal organelles and participating in a variety of essential cellular functions, such as signaling, electrolyte balance, and energy conversion. Cell membranes are structurally and chemically heterogeneous environment composed of numerous types of lipids arranged as a continuous bilayer. The assembly of protein complexes at the membrane surface is responsible for fundamental biological processes such as synaptic transmission, blood coagulation, and apoptosis. Resolving the macromolecular organization of these complexes at the membrane surface will help to understand the structural basis of their function and significance for the associated biological processes. In this review, we present our work on direct structure determination of membrane-bound clotting factors, specifically factor VIII (FVIII), by cryogenic electron microscopy (CryoEM). To resolve the FVIII membrane-bound organization, we have optimized lipid nanostructures resembling the activated platelet membrane. Combining structural CryoEM, capable of near-atomic resolution, with customized lipid nanotechnologies is a powerful approach to investigate how the cellular membrane can modulate protein function at close to physiological conditions. The outcome will open novel avenues for developing lipid nanotechnologies of diverse shapes and composition that can be optimized for various protein systems, germane for both drug delivery and macromolecular structure determination.

Nanodiscs (ND) are discoidal phospholipid bilayers stabilized by a pair of membrane-scaffolding proteins (MSP). The macromolecular composition and size of ND are ideal for structural and functional studies of membrane and membrane-associated proteins. In this work, we investigate the assembly of ND from a galactosylceramide and dioleoyl phosphatidylserine (PS) lipid mixture with two different MSP and at four MSP-to-lipid ratios. This lipid composition has been optimized for structural and biophysical studies of membrane-bound blood clotting factors that require Ca 2+ ions for function. We have demonstrated that CaCl 2 induces reversible stacking of the ND that depends on the ND size and Ca 2+ concentrations. Our biophysical and electron microscopy (EM) studies show a predominant ND population of ~12 nm in diameter for both the ND assembled from MSP1D1 to lipids ratio of 1:40 and from MSP1E3D1-to-lipids ratio of 1:80. Approximately half of the ND population assembled at MSP1E3D1-to-lipids ratio of 1:150 has a diameter of ~16 nm. These larger ND form ordered stacks at 5-m m Ca 2+ concentrations, as shown by cryo-EM. The number and length of the ND stacks increase with the increasing in Ca 2+ concentration. Adding millimolar concentrations of EDTA reverses the stacking of the ND.