Regular Article
Insertion and activation of functional Bacteriorhodopsin in a floating bilayer

https://doi.org/10.1016/j.jcis.2021.03.155Get rights and content

Abstract

The proton pump transmembrane protein bacteriorhodopsin was successfully incorporated into planar floating lipid bilayers in gel and fluid phases, by applying a detergent-mediated incorporation method. The method was optimized on single supported bilayers by using quartz crystal microbalance, atomic force and fluorescence microscopy techniques. Neutron and X-ray reflectometry were used on both single and floating bilayers with the aim of determining the structure and composition of this membrane-protein system before and after protein reconstitution at sub-nanometer resolution. Lipid bilayer integrity and protein activity were preserved upon the reconstitution process. Reversible structural modifications of the membrane, induced by the bacteriorhodopsin functional activity triggered by visible light, were observed and characterized at the nanoscale.

Introduction

2Supported lipid bilayers are often used as model membrane systems immobilized onto solid substrates and are capable of interacting with large molecules or molecular assemblies such as vesicles [1], nanoparticles [2], proteins, peptides and biopolymers [3]. Beyond their applications in biotechnology [4], they are important for many fundamental studies in physics (diffusion, mechanical properties, etc.) and biology (trafficking, endocytosis, exocytosis, membrane reshaping, etc.) [5], [6], [7], [8], [9] . In fact, by being an almost flat and immobilized system, they allow studies with high resolution techniques such as atomic force microscopy (AFM) [10], fluorescence resonance energy transfer (FRET) [11] and scattering of X-rays or neutrons [12]. One of the main drawbacks of adsorbed bilayers is that, due to their strong interaction with the solid substrate, they do not accurately replicate the conditions of natural membranes, which are inherently fluctuating systems and may enhance the activity of biomolecules such as membrane proteins. For this reason, polymer cushions have been used in the past as spacers between the substrate and the model membrane [13], [14]. Polymer cushions often induce significant roughness to the supported bilayer, which is not desirable for structural determinations. A more appropriate system for fine structural characterisation, developed by our team in recent decades, has involved the use of an adsorbed lipid bilayer acting as a spacer between a floating bilayer and the substrate [15]. This construct is often referred to as a double bilayer. A floating bilayer is more hydrated and free to fluctuate than an adsorbed bilayer [16], [17] and is particularly well-suited to investigate modifications in the bilayer physical properties induced by external stimuli (such as an electric field [18]), as well as bilayer interaction with nano-objects [19], [20] by means of surface scattering techniques. Double bilayers, as well as polymer cushioned lipid bilayers [21], are also promising candidates for the reconstitution of proteins in systems close to their natural environment, i.e. in conditions preserving the original protein activity [10]. The reconstitution process is not trivial. One of the challenges is related to the fragility of the floating layers that are weakly bound to the surface. Nevertheless, the insertion of active transmembrane proteins in floating bilayers opens up prominent perspectives for the study of fundamental biological processes at the molecular level [22], as well as the investigation into fascinating physical properties such as the fluctuations of an active membrane subjected to active athermal noise [23], [24].

In this work we aimed to develop a robust protocol for incorporation of bacteriorhodopsin (BR) while maintaining the structural integrity of the bilayers and the functional activity of the protein. Bacteriorhodopsin is a relatively small (27 kDa) and highly hydrophobic transmembrane protein composed of 248 amino acid residues [25], [26]. Importantly, it acts as a light-driven proton pump, where, upon absorption of a green light photon (λBR568 nm), it undergoes conformational changes and pumps a proton out of the cell [27], [28], [29]. Absorption and release of a proton are two simultaneous events during the bacteriorhodopsin photocycle. In nature, the protein can be found organised in 2D crystalline patches of trimers assembled within the so called purple membrane of the bacterium Halobacterium salinarum. The protein has shown stability in the purple membrane at a wide range of pH values (2–10) and temperatures (< 96°C) [30], [31]. BR was chosen for the large wealth of existing literature on its structure in the purple membrane and because its activation should lead to increased fluctuations that are detectable with the surface scattering methods developed by our team.

An incorporation method based on the use of sugar-based detergent n-dodecyl-β-D-maltopyranoside (DDM) was adapted from Refs [32], [33], [34] for inserting BR into floating bilayers. In order to characterize this insertion and demonstrate the activity of the protein, we have combined different experimental techniques. Quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy measurements enabled us to quantify the interactions between the protein and bilayers supported on surfaces, while AFM allowed us to obtain direct information on the insertion of proteins into the membrane. By using neutron (NR) and X-ray (XRR) reflectometry, we could access the structure of pristine bilayers and the structural and compositional modifications caused by protein incorporation and activation.

Double lipid bilayers are highly fragile systems, that need to be prepared by a combination of Langmuir–Blodgett and Langmuir-Schaefer (LB-LS) techniques. Appropriate preparation requires the use of relatively large substrates and the bilayers to be kept hydrated at all times, rendering their manipulation during QCM-D and AFM measurements near impossible. Consequently, we chose to optimize the incorporation protocol and confirm the insertion of the protein in single supported bilayers by using QCM-D and AFM techniques on supported bilayers prepared by vesicle fusion. Subsequently, we then demonstrated that the protocol could be applied to supported bilayers prepared by LB-LS via fluorescence microscopy, and studied the effect of BR insertion on scattering length density profiles by NR. This allowed us to validate the use of this technique for studying the insertion of bacteriorhodopsin. Finally, we investigated the insertion of BR in floating bilayers with fluorescence microscopy and neutron reflectivity, and demonstrated the activity of the protein with X-ray reflectivity experiments.

Section snippets

Materials

Synthetic zwitterionic phospholipids as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (purity >99%), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (purity >99%) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) (purity >99%) as well as n-dodecyl-β-D-maltopyranoside (DDM) (purity >99%) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD-PE) (purity >99%) were purchased from Avanti Polar Lipids (Lancaster, USA) and used

Results and discussion

The main goal of this work was to reconstitute bacteriorhodopsin into the floating bilayer of a double lipid bilayer system, while preserving its structure and function of bacteriorhodopsin upon insertion. One of the main challenges was the use of large substrates for double lipid bilayer formation for NR and XRR experiments (40 and 25 cm2). In addition, the preparation of double lipid bilayers required the implementation of adhoc bilayer deposition techniques for particular lipid composition.

Conclusions and perspectives

A robust and reproducible protocol for bacteriorhodopsin reconstitution into planar solid-supported single and floating phospholipid bilayers in gel and fluid phases was developed. A wide range of surface-sensitive techniques such as QCM-D, AFM, fluorescence microscopy, NR and XRR was used in order to confirm and optimize bacteriorhodopsin incorporation. Incubation time, protein and detergent concentration as well as lipid composition were optimized in order to preserve membrane structural

CRediT author statement

Tetiana Mukhina: Methodology, Validation, Formal analysis, Visualization, Investigation, Writing - Original Draft, Writing - Review and Editing. Yuri Gerelli: Methodology, Formal analysis, Software, Supervision, Investigation, Writing - Original Draft, Writing - Review and Editing, Resources. Arnaud Hemmerle: Investigation, Writing - Review and Editing. Alexandros Koutsioubas: Investigation. Kirill Kovalev: Resources. Jean-Marie Teulon: Formal analysis, Investigation. Jean-Luc Pellequer: Formal

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We acknowledge the Institut Laue-Langevin (ILL, Grenoble, France) (doi: 10.5291/ILL-DATA.8–02-803, doi: 10.5291/ILL-DATA.9–13-689), SOLEIL synchrotron (Saint-Aubin, France), JCNS-MLZ (Garching, Germany) for the beamtime allocations and technical support. We acknowledge the D17 team (ILL), Alessandro Coati and Benjamin Voisin (SixS, SOLEIL synchrotron, Saint-Aubin, France) and MARIA team (JCNS-MLZ, Garching, Germany) for assistance during the experiments. We are very grateful to Kalvin Buckley

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    Present address: Technischen Universität Darmstadt, Institut für Festkörperphysik, Hochschulstraße 8, 64289, Darmstadt, Germany

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