Метод визуализации и анализа Мембранные белков, взаимодействующих с помощью просвечивающей электронной микроскопии

In medical research, much attention is focused on membrane proteins, either intrinsic or extrinsic, involved in a variety of lipid interactions. Working with lipid-interacting proteins includes either selecting a substitute to the lipids, such as detergents, amphipols¹, or small proteins², or finding a membrane substitute that keeps the protein soluble and active. Lipoic membrane substitutes include liposomes and nanodiscs (ND)^3,4.

Nanodiscs are near-native membrane platforms developed by engineering the protein part, ApoA-1, of the high-density lipoprotein (HDL) naturally occurring in blood. ApoA-1 is a 243 residue-long chain of short amphipathic α-helices and has a lipid-free soluble conformation. In vitro when in the presence of lipids, two copies of the protein ApoA-1 spontaneously rearrange to encircle the hydrophobic acyl chain portion of a lipid bilayer patch⁵. Engineered versions of ApoA-1 are generally called membrane scaffolding proteins (MSP), and an increasing number are commercially available as plasmids or as purified proteins. Repetitions or deletions of the α-helices in ApoA-1 result in longer⁶ or shorter⁷ membrane scaffolding proteins. This in turn makes it possible to form discs around 6 nm⁷ to 17 nm⁸ in diameter. There are different types of applications for the nanodiscs^3,9. The most commonly used application is to provide a near-native membrane environment for the stabilization of an integral membrane protein⁸, reviewed previously^3,9. A less-explored use is to provide a nanoscale membrane surface for the study of peripheral membrane proteins^{10,11,12,13,14,15,16,17}. Section 1 of the protocol below visualizes the procedure for making nanodiscs composed of phospholipids and membrane scaffolding protein.

Sample preparation is a bottleneck in most methods. Method-specific samples may add particular information, but they also make comparisons of results difficult. Therefore, it is simpler when samples are multimodal and can be used directly in several different methods. One advantage with the use of nanodiscs is the small size of the nanodisc in comparison to liposomes (e.g., the samples can be directly used for both TEM and non-denaturing gel electrophoresis, as in the present protocol).

Vesicles and liposomes have long been used to understand the function of membrane-interacting proteins. For structural studies and visualization, an example of the structural determination of a transmembrane protein in liposomes is available¹⁸. However, no high-resolution 3D structure of a monotopic membrane protein embedded on a liposome membrane has been published yet, as far as we know. Gold nanoparticles or antibodies can be used to visualize proteins binding to liposomes or vesicles using TEM¹⁹. Even though these probes are very specific, they might interfere with membrane-binding proteins by veiling the membrane binding site or by masking areas of interest with the flexible parts. Gold-labeled or antibody-complexed proteins could probably be analyzed on a gel, but this would increase the cost of the experiment.

Though liposomes are an excellent platform, one cannot be certain that the population has a particular ratio of protein per liposome, a feature that can be explored by the use of nanodiscs²⁰. In a liposome, cofactors and substrates can be trapped in the soluble interior. Substances that are membrane-soluble will share the same fate for both types of membrane mimetics. Nevertheless, as the bilayer area is smaller in nanodiscs, a smaller amount of substance is required to saturate the nanodisc membranes.

Understanding protein function through the determination of the atomic structure has been essential for many fields of research. Methods for protein structure determination include X-ray²¹; nuclear magnetic resonance (NMR)^22,23; and transmission electron microscopy (TEM)²⁴ at cryogenic temperatures, cryoEM. The resolution by cryoEM has lately been greatly improved, mainly due to the use of direct electron detectors^25,26. The macromolecules are imaged in thin, vitreous ice²⁷ in a near-native state. However, due to the low contrast of biological molecules, they become hard to detect in the size range of 100 – 200 kDa. For suitably sized samples, data collection can be made and the method of single particle reconstruction can be applied to obtain a structure²⁸.

However, the determination of protein structure by TEM is a multistep process. It usually starts with the evaluation of sample monodispersity by negative-stain TEM²⁹ using salts of heavy metals like phosphotungsten (PT)³⁰ or uranium³¹. Reconstruction of a low-resolution model of the negatively stained macromolecule is usually made and may yield important information on the molecular structure²⁹. In parallel, data collection using cryoEM may start. Care should be taken when evaluating negative-stain TEM data to avoid the misinterpretation of artefact formation. One particular artefact is the effect of the PT stain on phospholipids and liposomes³², resulting in the formation of long rods resembling stacks of coins viewed from the side³³. Such "rouleau" or "stacks" (hereafter denoted as "stacks") were observed early on for HDL³⁴, and later also for nanodiscs³⁵.

The stacking and reshaping of membranes may occur for many reasons. For example, it can be induced by co-factors like copper, shown by TEM imaging in an ammonium molybdate stain³⁶. A fraction of the membrane lipids in liposomes contained an iminodiacetic acid head group mimicking metal complexation by EDTA, thus stacking liposomes after the addition of copper ions³⁶. Stacking could also be due to a protein-protein interaction by a protein in or on the lipid bilayers (the stain used is not mentioned)³⁷. The stack formation of phospholipids by PT was observed early on; however, later work has focused on removing or abolishing this artifact formation³⁸.

Here, we propose a method to take advantage of the NaPT-induced nanodisc stacking for the study of membrane-binding proteins by TEM. In short, protein binding on the nanodiscs would prevent the nanodiscs from stacking. Though the reasons for the stacking are not clear, it was proposed³⁹ that there is an electrostatic interaction between the phospholipids and the phosphoryl group of PT, causing the discs to stick to each other (Figure 1A). The hypothesis behind our protocol is that when a protein binds to a nanodisc, most of the phospholipid surface is not available for the interaction with the PT due to steric hindrance by the protein. This would prevent stack formation (Figure 1B). Two conclusions can be drawn. First, the prevention of stacking means that the protein of interest has bound to the membrane. Secondly, the protein-ND complex can be treated with standard single-particle processing methods^24,40 to get a rough morphology of the complex. Furthermore, analyses by methods like non-denaturing gel electrophoresis or dynamic light scattering can be performed.

To demonstrate this hypothesis, we used the membrane-binding protein 5-lipoxygenase (5LO), which is involved in many inflammatory diseases^41,42. This 78-kDa protein requires calcium ions to bind to its membrane⁴³. Though this membrane association has been studied extensively using liposomes^44,45,46 and membrane fractions⁴⁷, these cannot be used for TEM analysis and structure determination.

The preparation of nanodiscs starts by mixing MSP with lipid resuspended in the detergent sodium cholate. After incubation on ice for 1 h, the detergent is slowly removed from the reconstitution mixture using an adsorbent resin. This kind of material is frequently made of polystyrene shaped into small beads. They are relatively hydrophobic and have a strong preference for binding detergent compared to lipids⁴⁸. After removing the hydrophobic beads and performing clarification using centrifugation, the nanodiscs are purified by size exclusion chromatography (SEC). The purified nanodiscs are mixed with a monotopic membrane protein (and possible cofactors) in an equimolar ratio (or several ratios for a titration) and are left to react (15 min). Analysis by TEM is carried out by applying µL-amounts of sample onto glow-discharged, carbon-coated grids and then by performing negative staining with NaPT. The same sample from when the aliquots were applied to the TEM grids can be used for analysis by non-denaturing or SDS PAGE gel-electrophoresis, as well as by various kinds of activity measurements, with no major changes.