Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

Jelmer Coenradij; Eleonora Bailoni; Bert Poolman

doi:10.3791/66627

JoVE Journal > Biochemistry

생화학

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles

Published: April 12, 2024

doi:

10.3791/66627

Jelmer Coenradij, Eleonora Bailoni, Bert Poolman

¹Department of Biochemistry,University of Groningen

Summary

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

Abstract

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy.

Introduction

The field of bottom-up synthetic biology focuses on constructing (minimal) cells¹^,² and metabolic bioreactors for biotechnological³^,⁴ or biomedical purposes⁵^,⁶^,⁷^,⁸. The construction of synthetic cells provides a unique platform that allows researchers to study (membrane) proteins in well-defined conditions mimicking those of native environments, enabling the discovery of emergent properties and concealed biochemical functions of proteins and reaction networks⁹. As an intermediate step towards an autonomously functioning synthetic cell, modules are developed that capture essential features of living cells such as metabolic energy conservation, protein and lipid synthesis, and homeostasis. Such modules not only enhance our understanding of life but also have potential applications in the fields of medicine⁸ and biotechnology¹⁰.

Transmembrane proteins are at the heart of virtually any metabolic network as they transport molecules in or out of the cell, signal, and respond to the quality of the environment, and play numerous biosynthetic roles. Thus, the engineering of metabolic modules in synthetic cells requires in most cases the reconstitution of integral and/or peripheral membrane proteins into a membrane bilayer composed of specific lipids and high integrity (low permeability). The handling of these membrane proteins is challenging and requires specific knowledge and experimental skills.

Several methods have been developed to reconstitute membrane proteins within phospholipid vesicles, most often with the purpose of studying the function¹¹^,¹², regulation¹³, kinetic properties¹⁴^,¹⁵, lipid dependence¹⁵^,¹⁶, and/or stability¹⁷ of a specific protein. These methods involve the rapid dilution of detergent-solubilized protein into aqueous media in the presence of lipids¹⁸, the removal of detergents by incubating detergent-solubilized protein with detergent-destabilized lipid vesicles and absorption of the detergent(s) onto polystyrene beads¹⁹, or the removal of detergents by dialysis or size-exclusion chromatography²⁰. Organic solvents have been used to form lipid vesicles, for example, via the formation of oil-water interphases²¹, but the majority of integral membrane proteins are inactivated when exposed to such solvents.

In our laboratory, we mostly reconstitute membrane proteins by the detergent-absorption method to form large-unilamellar vesicles (LUVs)¹⁹. This method allows the co-reconstitution of multiple membrane proteins and the encapsulation in the vesicle lumen of enzymes, metabolites, and probes²²^,²³. The membrane protein-containing LUVs can be converted into giant-unilamellar vesicles (GUVs) with/without encapsulation of water-soluble components, using either electroformation²⁴ or gel-assisted swelling²⁵ and specific conditions to preserve the integrity of the membrane proteins²⁶.

This paper presents a protocol for the reconstitution in LUVs of an out-of-equilibrium metabolic network that regenerates ATP through the breakdown of L-arginine into L-ornithine²⁷. The formation of ATP is coupled to the production of glycerol-3-phosphate (G3P), an important building block for phospholipid synthesis²²^,²⁸. The metabolic pathway consists of two integral membrane proteins, an arginine/ornithine (ArcD) and a G3P/Pi antiporter (GlpT). In addition, three soluble enzymes (ArcA, ArcB, ArcC) are required for the recycling of ATP, and GlpK is used to convert glycerol into glycerol 3-phosphate, using the ATP from the breakdown of L-arginine, see Figure 1 for a schematic overview of the pathway. This protocol represents a good starting point for the future construction of even more complex reaction networks-for the synthesis of lipids or proteins or the division of cells. The lipid composition of the vesicles supports the activity of a wide variety of integral membrane proteins and has been optimized for the transport of diverse molecules into or out of the vesicles²⁷^,²⁹^,³⁰.

Figure 1: Overview of the pathway for ATP production and glycerol 3-phosphate synthesis and excretion. Please click here to view a larger version of this figure.

In short, purified membrane proteins (solubilized in dodecyl-β-D-maltoside, DDM) are added to preformed lipid vesicles that have been destabilized with Triton X-100, which allows the insertion of the proteins into the membrane. The detergent molecules are subsequently (slowly) removed by the addition of activated polystyrene beads, resulting in the formation of well-sealed proteoliposomes. Soluble components can then be added to the vesicles and encapsulated via freeze-thaw cycles, which traps the molecules in the process of membrane fusion. The obtained vesicles are highly heterogeneous and many are multilamellar. They are then extruded through a polycarbonate filter with a pore size of 400, 200, or 100 nm, which yields more uniformly sized vesicles; the smaller the pore size, the more homogeneous and unilamellar the vesicles but at the price of a smaller internal volume. Non-incorporated proteins and small molecules are removed from the external solution by size-exclusion chromatography. The proteoLUVs can be converted into micrometer size vesicles by gel-assisted swelling, and these proteoGUVs are then collected and trapped in a microfluidic chip for microscopic characterization and manipulation. Figure 2 shows a schematic overview of the full protocol.

Figure 2: Overview of the protocol for reconstituting membrane proteins and encapsulating enzymes and water-soluble components in lipid vesicles of sub-micrometer (LUVs) and micrometer size (GUVs). Please click here to view a larger version of this figure.

The reconstitution and encapsulation protocols work well and the functionality of the proteins is retained, but the proteoLUVs and proteoGUVs are heterogeneous in size. Microfluidic approaches³¹^,³² allow the formation of micrometer-sized vesicles that are more homogeneous in size, but functional reconstitution of membrane proteins is generally not possible because residual solvent in the bilayer inactivates the proteins. The proteoLUVs range in size from 100 to 400 nm, and at low concentrations of enzymes, the encapsulation may lead to vesicles with incomplete metabolic pathways (stochastic effects; see Figure 3). LUVs are ideal for constructing specific metabolic modules, as shown here for the production of ATP and building blocks like G3P. Such proteoLUVs can potentially be encapsulated in GUVs and serve as organelle-like compartments for the host vesicles.

Figure 3: Number of molecules per vesicle with a diameter of 100, 200, or 400 nm. (A) When the encapsulated proteins (enzymes, probes) are in the range of 1-10 µM. (B) The reconstitution is done at 1 to 1,000, 1 to 10,000, and 1 to 100,000 membrane proteins per lipid (mol/mol). We make the assumption that molecules are encapsulated at the indicated concentrations and incorporated in the membrane at these protein-to-lipid ratios. For some enzymes, we have seen that they bind to membranes, which can increase their apparent concentration in the vesicles. Abbreviation: LPR = Lipid-Protein-Ratio Please click here to view a larger version of this figure.

Protocol

1. General preparation Chemicals Dissolve lipids (in powder form) to 25 mg/mL in CHCl3 for making preformed liposomes. NOTE: It is preferable to prepare fresh lipid stocks, but the stock solutions can also be stored at -20 °C for a few weeks. Working with lipids in powder form is more accurate than using lipids already solubilized in CHCl3. CHCl3 should be handled using glass pipettes and/or syringes and stored in glass containers as C…

Representative Results

The reconstitution of solubilized membrane proteins in liposomes requires the destabilization of preformed vesicles. The addition of low amounts of Triton X-100 initially results in an increase of absorbance at 540 nm (A540) due to an increase in light scattering by the swelling of the vesicles (Figure 4). The maximum A540 value is the point where the liposomes are saturated with detergent (Rsat), after which any further addition of Triton X-100 will…

Discussion

We present a protocol for the synthesis of (membrane) protein containing sub-micrometer size lipid vesicles (proteoLUVs), and the conversion of proteoLUVs into giant-unilamellar vesicles (proteoGUVs). The protocol should be applicable for the reconstitution of other membrane proteins¹³^,¹⁹^,³⁰^,⁴⁰ and the encapsulation of metabolic networks other than the L-arginine breakdown and glycerol 3-phosph…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Aditya Iyer for the cloning of the pBAD-PercevalHR gene and Gea Schuurman-Wolters for aiding with protein production and purification. The research was funded by the NWO Gravitation program "Building a Synthetic Cell" (BaSyC).

Materials

Agarose	Sigma Aldrich	A9414-25g
Amicon cut-off filter	Sigma Aldrich	Milipore centrifugal filter units Amicon Ultra
BioBeads	BioRad	152-3920
CHCl₃	Macron Fine Chemicals	MFCD00000826
D(+)-Glucose	Formedium	–
D(+)-Sucrose	Formedium	–
DDM	Glycon	D97002 -C
Diethyl Ether	Biosolve	52805
DMSO	Sigma-Aldrich	276855-100ml
DOPC	Avanti	850375P-1g
DOPE	Avanti	850725P-1g
DOPG	Avanti	840475P-1g
DTT	Formedium	DTT005
EtOH	J.T.Baker Avantor	MFCD00003568
Extruder	Avestin Inc	LF-1
Fluorimeter	Jasco	Spectrofluorometer FP-8300
Glycerol	BOOM	51171608
Gravity flow column	Bio-Rad	732-1010
Hamilton syringe 100 µL	Hamilton	7656-01
Hamilton syringe 1000 µL	Hamilton	81320
Handheld LCP dispenser	Art Robbins Instruments	620-411-00
Handheld Sonicator	Hielscher Ultrasound Technology	UP50H
HCl	BOOM	x76021889.1000
Imidazole	Roth	X998.4-250g
K₂HPO₄	Supelco	1.05099.1000
KCl	BOOM	76028270.1
KH₂PO₄	Supelco	1.04873.1000
Kimwipe	Kimtech Science	7552
Large Falcon tube centrifuge	Eppendorf	Centrifuge 5810 R
L-Arginine	Sigma-Aldrich	A5006-100G
Light microscope	Leica	DM LS2
L-Ornithine	Roth	T204.1
LSM Laser Scanning Confocal Microscope	Zeiss	LSM 710 ConfoCor 3
MgCl₂	Sigma-Aldrich	M2670-1KG
Microfluidic chip	Homemade	PDMS based	DOI: https://doi.org/10.1039/C8LC01275J
Na-ADP	Sigma-Aldrich	A2754-1G
NaCl	Supelco	1.06404.1000
Nanodrop Spectrometer	Isogen Life Science	ND-1000 spectrophotometer NanoDrop
NaOH	Supelco	1.06498.1000
Needles for GUVs	Henke-Ject	14-14575	27 G x 3/4'' 0.4 x 20 mm
Needles for microfluidics	Henke-Ject	14-15538	18 G x 1 1/2'' 1.2 x 40 mm
Ni₂⁺ Sepharose	Cytiva	17526802
Nigericin	Sigma-Aldrich	N7143-5MG
Nutator	VWR	83007-210
Osmolality meter	Gonotec Salmenkipp	Osmomat 3000 basic freezing point osmometer
Plasmacleaner	Plasma Etch	PE-Avenger
Polycarbonate filter	Cytiva Whatman	Nuclepor Track-Etch Membrane Product: 10417104	0.4 µm
Polycarbonate ultracentrifuge tube	Beckman Coulter	355647
Pyranine	Acros Organics	H1529-1G
Quartz cuvette (black)	Hellma Analytics	108B-10-40
Sephadex G-75 resin	GE Healthcare	17-0050-01
Sonicator	Sonics Sonics & Materials INC	Sonics vibra cell
Syringe filter	Sarstedt	Filtropur S plus 0.2	0.2 µm
Syringe pump	Harvard Apparatus	A-42467
Tabletop centrifuge	Eppendorf	centrifuge 5418
Teflon spacer	Homemade	Teflon based	45 x 26 x 1.5 or 45 x 26 x 3 or 20 x 20 x 3 mm
Tris	PanReac AppliChem	A1086.1000
Triton X-100	Sigma Aldrich	T8787-100 ml
Ultracentrifuge	Beckman Coulter	Optima Max-E
UV lamp	Spectroline	ENB-280C/FE
UV/VIS Spectrometer	Jasco	V730 spectrophotometer
Valinomycin	Sigma-Aldrich	V0627-10MG
Widefield fluorescence microscope	Zeiss	AxioObserver
β-Casein	Sigma Aldrich	C5890-500g

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Coenradij, J., Bailoni, E., Poolman, B. Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles. J. Vis. Exp. (206), e66627, doi:10.3791/66627 (2024).