Articles sur le même thème :
Membrane proteins (MPs) are very important in cells as they accomplish a diversity of roles, justifying the increasing interest in their study.
However, functional studies of MPs within a native cellular setting are difficult to perform due to the innate complexity of the cell membrane. To surpass these difficulties, MPs can be purified from their native environment and reinserted into membrane mimetic systems, such as liposomes, enabling their functional study. The resulting proteoliposomes are, currently, essential experimental systems for the mechanistic understanding of MPs.
Proteoliposomes have been used to investigate membrane transport, study membrane fusion events (such as virus-host cell interactions), assess NMR-based structure determination of MPs in their proper folding, and have also been used as promising drug delivery systems for both hydrophilic and hydrophobic drugs [1].
At this moment, functional measurements are becoming the rate-limiting step in the study of MPs using liposomes and several challenges are presented, namely the difficulty of successful reconstitution, co-reconstitution and controlled orientation of MPs into liposomes, and the development of improved methods for the precise characterization of MPs in proteoliposomes and for reaction monitoring, namely the development of fluorescent dyes with high sensitivity and high temporal resolution [1].
Strategies for Effective Reconstitution of Membrane Proteins in Liposomes
The effective reconstitution of MPs in liposomes (which directly affects their activity) is highly influenced by the lipid composition of liposomes. Differently from native membranes, proteoliposomes contain much fewer proteins since higher amounts of proteins frequently impair the reconstitution process.
Several strategies have been developed to overcome this limitation, such as the GRecon method that uses a sucrose density gradient with increasing concentrations of cyclodextrin and detergent-destabilized liposomes. During the reconstitution process, the detergent is gradually replaced by lipids, yielding proteoliposomes with high protein content [2].
MPs have also been embedded in small lipid bilayer discs surrounded by a scaffold protein mimicking the properties of nanodiscs. The advantage of these systems is that their size can be modulated by varying the peptide to lipid ratio, but they require the prior use of detergents to extract the protein from the native membrane [3]. To avoid the use of detergent, styrene-maleic acid (SMA) lipid particles (SMALPs) can be successfully employed to purify and functionally reconstitute MPs into lipid bilayers [4].
Controlling Membrane Protein Orientation in Proteoliposomes
It is known that the relative orientation of the inserted MPs significantly affects functional studies performed with proteoliposomes. However, the control of MP orientation in liposomes is difficult to achieve because, during a reconstitution process, MPs are inserted in a random orientation, resulting in the production of different proteoliposome populations and causing heterogeneity in the experimental system [1].
Orientation seems to be more uniform when the MPs are reconstituted into preformed and partially detergent-solubilized liposomes [5]. Other approaches employed for guided orientation include the use of Ni-NTA-functionalized beads to immobilize His-tagged MPs prior to reconstitution [6], and the attachment of a fusion domain on either end of the protein, which allows to chose one of the two possible orientations [7].
Although it seems to work for small proteins with no soluble domain, it remains to be tested with large MPs. Moreover, there is also the need for robust and easy-to-implement methods for the determination of protein relative orientation that will allow to evaluate new reconstitution methods [1].
Co-reconstitution of Multiple Membrane Proteins in Liposomes
Co-reconstitution of MPs, i.e., the incorporation of different MPs into the same liposome is desirable because it enables to study the interplay of different proteins at the molecular level and to perform functional measurements comparing kinetics and efficiencies between individual or multiple complexes [1].
Given the individual requirements of each MP for optimal reconstitution and orientation, this is difficult to attain, and it is proposed that it should be carried out in two steps, namely individual reconstitution of each protein under optimal conditions followed by fusion of both populations.
This has not yet been tested with MPs-containing liposomes [1]. Alternatively, a method that ensures a 1:1 reconstitution stoichiometry of MPs has been developed, based on maleimide chemistry and the use of DNA linkers with appropriate lengths [8].
Designing Fluorescent Probes and Utilizing Giant Unilamellar Vesicles in Proteoliposome Research
Another challenge in proteoliposome research relates with the rational design of fluorescent dyes to follow protein function. In this regard, the use of membrane-anchored fluorescent probes offers advantages over soluble dyes as the former are efficiently and stably incorporated into the liposomes and do not leak from the membrane.
Their main drawback is that they are randomly distributed in both leaflets of the bilayer. To surpass this problem, a DNA double strand may be used between the lipophilic anchor and the fluorescent moiety [9]. More advanced membrane-anchored probes rely on structural changes of environmentally sensitive DNA motifs [10]. Although these strategies have been mostly used in vivo, these complex probes could be useful for liposomal studies.
Single-molecule experiments with liposome-embedded MPs could also enable to monitor binding change mechanisms and pH change within the lumen of small unilamellar vesicles (SUVs) [11].
Future directions of proteoliposome research encompass the use of giant unilamellar vesicles (GUVs) to study MP function [1]. GUVs are much larger than convention liposomes and, thus, can be directly observed by light microscopy techniques. Besides, their increased surface and inner volume allows for the incorporation of entire protein machineries, small vesicles or even whole bacteria, mimicking complex functions of cells, and showing potential in the research on artificial cells. However, they are less robust than classical liposomes and their preparation is less straightforward. In addition, the insertion of MPs into the GUV membrane is not as easy, which has impaired, so far, their use in vectorial transport experiments with MPs [12].
Furthermore, cell-sized vesicles formed from synthetic block polymers, called polymersomes, have been successfully used to mimic compartmentalization of the eukaryotic cell or to enable spatial separation of multienzyme synthesis reactions [13].
Moreover, detection methods with high temporal resolution and sensitivity are required, as well as photostable fluorophores to reduce bleaching during observation. Some of the current approaches such as single-molecule measurements and microfluidics demand for technical expertise and specialized equipments. Finally, as microscopy experiments produce a large amount of data, user-friendly yet powerful analytical software is also necessary [1].
Written by Luísa Silva, PhD & Scientific writer.
Synthelis
As a service provider with more than 13 years of experience in cell-free technology and membrane protein expression, Synthelis can produce functional and oriented membrane proteins in proteoliposome format using either its patented approach to directly incorporate the protein target into the lipid bilayer of the liposomes, or after protein solubilization and purification followed by a relipidation phase. Both approaches have their pros and cons. Our team has also the know-how and the experience to produce membrane proteins either solubilized in detergent or in nanodisc format.
If you have such project and want to discuss its feasibility, please contact us.
Sources
[1] Amati A.M., Graf S., Deutschmann S., Dolder N., von Ballmoos C. 2020. Current problems and future avenues in proteoliposome research. Biochem. Soc. Trans. 48:1473-1492.
https://doi.org/10.1042/BST20190966
[2] Althoff T., Davies K.M., Schulze S., Joos F., Kühlbrandt W. 2012. GRecon: A method for the lipid reconstitution of membrane proteins. Angew. Chem. Int. Ed. Engl. 51:8343-8347.
https://doi.org/10.1002/anie.201202094
[3] Schuler M.A., Denisov I.G., Sligar S.G. 2013. Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol. Biol. 974:415-433. https://doi.org/10.1007/978-1-62703-275-9_18
[4] Dörr J.M., Scheidelaar S., Koorengevel M.C., Dominguez J.J., Schäfer M., van Walree C.A., Killian J.A. 2016. The styrene–maleic acid copolymer: a versatile tool in membrane research. Eur. Biophys. J. 45:3-21. https://doi.org/10.1007/s00249-015-1093-y
[5] Pagano A. Spiess M. 2005. Reconstitution of Rab4-dependent vesicle formation in vitro. Methods Enzymol. 403:81-92. https://doi.org/10.1016/S0076-6879(05)03008-9
[6] Zheng H., Lee S., Llaguno M.C., Jiang Q.-X. 2016. bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles. J. Gen. Physiol. 147:77-93. https://doi.org/10.1085/jgp.201511448
[7] Ritzmann N., Thoma J., Hirschi S., Kalbermatter D., Fotiadis D., Müller D.J. 2017. Fusion domains guide the oriented insertion of light-driven proton pumps into liposomes. Biophys. J. 113:1181-1186.
https://doi.org/10.1016/j.bpj.2017.06.022
[8] Raschle T., Lin C., Jungmann R., Shih W.M., Wagner G. 2015. Controlled co-reconstitution of multiple membrane proteins in lipid bilayer nanodiscs using DNA as a scaffold. ACS Chem. Biol. 10:2448-2454.
https://doi.org/10.1021/acschembio.5b00627
[9] Dolder N., von Ballmoos C. 2020. Bifunctional DNA duplexes permit efficient incorporation of pH probes into liposomes. ChemBioChem. 21:2219-2224. https://doi.org/10.1002/cbic.202000146
[10] Dembska A., Bielecka P., Juskowiak B. 2017. pH-Sensing fluorescence oligonucleotide probes based on an i-motif scaffold: a review. Anal. Methods 9:6092-6106. https://doi.org/10.1039/c7ay01942d
[11] Veshaguri S., Christensen S.M., Kemmer G.C., Ghale G., Møller M.P., Lohr C., Christensen A.L., Justesen B.H., Jørgensen I.L., Schiller J., Hatzakis N.S., Grabe M., Pomorski T.G., Stamou D. 2016. Direct observation of proton pumping by a eukaryotic P-type ATPase. Science 351:1469-1473.
https://doi.org/10.1126/science.aad6429
[12] Jørgensen I.L., Kemmer G.C., Pomorski T.G. 2017. Membrane protein reconstitution into giant unilamellar vesicles: a review on current techniques. Eur. Biophys. J. 46:103-119.
https://doi.org/10.1007/s00249-016-1155-9
[13] Klermund L., Poschenrieder S.T., Castiglione K. 2017. Biocatalysis in polymersomes: improving multienzyme cascades with incompatible reaction steps by compartmentalization. ACS Catal. 7:3900-3904. https://doi.org/10.1021/acscatal.7b00776