The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1. Scaffold Liposome Preparation
NOTE: Ideally, initial experiments should use a relatively simple and featureless scaffold (comprised of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine or PC) and DGS-NTA(Ni2+) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt)). Building off of these experiments, lipid charge, flexibility, and curvature can be introduced as individual factors with the potential to alter membrane-proximal interactions. These changes can be achieved by adding defined amounts of specific lipid constituents, including phosphatidylserine or cardiolipin (CL), phosphatidylethanolamine (DOPE or PE), or galactosyl(&#946;) ceramide.
<ol>
	<li>Combine lipids dissolved in chloroform in a clean glass test tube. Evaporate the solvent with dry nitrogen gas while rotating the tube to form a thin lipid film. Remove residual solvent with a centrifugal evaporator for 1 h at 37 &#176;C. 
	NOTE: Various liposome formulations are used in the protocols described below: scaffold liposomes (3.3 mol% DGS-NTA(Ni2+) / 96.7 mol% DOPC), scaffold liposomes with cardiolipin (3.3 mol% DGS-NTA(Ni2+) / 10 mol% cardiolipin / 86.7 mol% DOPC), flexible scaffold liposomes with cardiolipin (3.3 mol% DGS-NTA(Ni2+) / 10 mol% cardiolipin / 35 mol% DOPE / 51.7 mol% DOPC), and enriched scaffold liposomes (10 mol% DGS-NTA(Ni2+) / 15 mol% cardiolipin / 35 mol% DOPE / 40 mol% DOPC).</li>
	<li>Add Buffer A (25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM KCl, pH adjusted to 7.5 with KOH) preheated to 37 &#176;C such that the final lipid concentration is 1 - 2 mM. Incubate 30 min at 37 &#176;C with occasional vortexing to fully resuspend the lipid mixture (Figure 1a).</li>
	<li>Transfer to a plastic test tube, place the tube in liquid nitrogen until completely frozen (roughly 30 s), and place in a 37 &#176;C water bath until fully thawed (roughly 1 - 2 min). Repeat for a total of 4 freeze-thaw cycles (Figure 1b).</li>
	<li>Prepare a lipid extruder by soaking 4 filter supports and a polycarbonate filter in buffer and assembling the extruder according to manufacturer instructions. Extrude the lipid solution through the filter 21 times. Use gentle, constant pressure to ensure a homogenous size distribution (Figure 1c). 
	NOTE: For all experiments described in this protocol, a 1.0 &#181;m polycarbonate filter was used for extrusion. Drp1 interaction with anionic lipids can be observed with a variety of liposome diameters ranging from 50 nm to 400 nm12 or larger13. Hence, the filter size of 1 &#181;m was chosen to be ideal for both GTPase activity and for electron microscopy. If other liposome diameters are desired, preparation of giant unilamellar vesicles14,15 (GUVs) or small unilamellar vesicles16 (SUVs) can be used. Dynamic light scattering can be used to assess liposome size heterogeneity13.</li>
	<li>Store extruded liposomes at 4 &#176;C and discard after 3 - 5 d.</li>
</ol>
2. Use of Scaffold Liposomes for Protein Binding Analysis
<ol>
	<li>Sample Preparation
	<ol>
		<li>Incubate His-tagged Mff&#916;TM (5 &#181;M final) with scaffold liposomes (40 mol% PC / 35 mol% PE / 15 mol% CL / 10 mol% DGS-NTA(Ni2+); 50 &#181;M final) for at least 15 min at RT in Buffer A + BME (25 mM HEPES, 150 mM KCl, 10 mM &#946;-mercaptoethanol (BME), pH adjusted to 7.5 with KOH). For an Mff-free control, incubate liposomes with a his-tagged control protein (such as GFP) to bind and shield exposed NTA(Ni2+). 
		NOTE: Mff&#916;TM was expressed and purified as described in a previous study2. GFP was purified in a similar manner, but the ion-exchange step was omitted. BME was required for these experiments because Drp1 is sensitive to oxidation, which can alter its activity and assembly properties.</li>
		<li>Add Drp1 (2 &#181;M final) and incubate for 1 h at RT. 
		NOTE: Drp1 was expressed and purified as described in a previous study2. After incubation with Drp1, the effect of nucleotide binding on membrane deformation can be investigated by incubating one additional hour with 2 mM MgCl2 and either 1 mM GTP, 1 mM GMP-PCP, or Buffer A + BME.</li>
	</ol>
	</li>
	<li>Negative Stain Transmission Electron Microscopy (EM) Analysis
	<ol>
		<li>Transfer 5 &#181;L of sample to a sheet of laboratory film, and lay a carbon-coated Cu/Rh grid on the sample. Incubate the grid 1 min on the sample, blot away excess liquid on filter paper, and transfer to a drop of 2% uranyl acetate. Incubate 1 min, blot excess stain on filter paper, and transfer to a grid box. Store under vacuum O/N to ensure full desiccation.</li>
		<li>Image samples using a transmission electron microscope at 18,500 - 30,000X&#160;magnification to observe ultrastructural changes in protein and liposome morphologies17.&#160; 
		Note: Ultrastructural changes can be quantified using image analysis software, such as ImageJ13 (http://imagej.nih.gov/ij/). Protein decoration can be measured when compared with naked lipid templates. Additionally, the diameters of tubular segments can be measured from the outermost portion of the assemblies13. A more detailed analysis can be performed using cryo-electron microscopy17. This method can be used to image native protein-lipid assemblies in solvent without the use of heavy metal stains that coat the sample. In this way, detailed structural features not apparent in negative stain, including changes in the underlying lipid morphology, can be examined and quantified.</li>
	</ol>
	</li>
</ol>
3. Use of Scaffold Liposomes for Enzymatic Assay
Note: A colorimetric GTPase assay18 was used to measure phosphate liberation via GTP hydrolysis. Alternative GTPase assays are available19 and can be implemented as needed.
<ol>
	<li>Incubate His-tagged Mff&#916;TM (Mff), Fis1&#916;TM(Fis1), or GFP (5 &#181;M final for all) with scaffold liposomes (150 &#181;M final) for 15 min at RT in Buffer A + BME (volume = 30 &#181;L). Add Drp1 (500 nM final) and incubate an additional 15 min at RT (volume = 80 &#181;L). 
	NOTE: Fis1 was purified in a similar manner to Mff2, but the ion-exchange chromatography step was omitted. The purpose of His-tagged GFP is to shield the NTA(Ni2+) headgroups and prevent nonspecific charge interactions with other proteins. If no effect is observed in the absence of GFP, then this control may not be required. Alternative blocking proteins (of comparable size to the protein of interest) can be used as well, but GFP allows for direct visualization of the interactions with scaffold liposomes.</li>
	<li>Transfer tubes to a thermocycler set to 37 &#176;C, and initiate reactions by addition of GTP and MgCl2 (1 mM and 2 mM final, respectively; volume = 120 &#181;L).</li>
	<li>At desired time points (i.e. T = 5, 10, 20, 40, 60 min), transfer 20 &#181;L of reaction to wells of a microtiter plate containing 5 &#181;L of 0.5 M EDTA to chelate Mg2+ and stop the reaction.</li>
	<li>Prepare a set of phosphate standards by diluting KH2PO4 in Buffer A + BME to calibrate results. A useful set of standards is 100, 80, 60, 40, 20, 10, 5, and 0 &#181;M. Add 20 &#181;L of each to wells containing 5 &#181;L of 0.5 M EDTA.</li>
	<li>Add 150 &#181;L of Malachite green reagent (1 mM malachite green carbinol, 10 mM ammonium molybdate tetrahydrate, and 1 N HCl) to each well, and read OD650 5 min after addition. 
	NOTE: GTP is acid labile, and will hydrolyze in the presence of malachite green reagent. Ensure that the time between adding malachite reagent and reading is constant to ensure reproducible results.</li>
	<li>Generate a standard curve by plotting OD650 of the standards as a function of phosphate concentration. Use linear regression to determine the relationship between OD650 and phosphate concentration in a sample.</li>
	<li>Using the linear regression, convert the OD650 of the protein reaction samples to &#181;M phosphate. Determine the rate of phosphate generation for each reaction mixture by plotting phosphate concentration as a function of time, and convert to kcat by dividing the rate by the Drp1 concentration (0.5 &#181;M). 
	NOTE: Only the initial linear rate should be used to determine the rate of phosphate generation, and a minimum of 3 data points must be used. If the rate of reaction is sufficiently rapid that the first three data points are not linear (i.e. the r2 of the linear fit is less than 0.9) a significantly shorter time course with at least 3 time points should be performed.</li>
</ol>

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The authors have nothing to disclose.

This protocol offers a method for investigating protein-protein interactions involving integral membrane proteins. By utilizing a modular liposome scaffold, investigators are capable of assessing the activity of one or more proteins in a lipid-proximal environment. Previous studies have demonstrated a similar method for receptor enzymes of the plasma membrane24-26. We have expanded this method to incorporate lipid cofactors and explore interactions between proteins that make up the mechanoenzymatic core of the mitochondrial fission machinery.
For the model system presented above, we found that Mff-decorated SL enhanced Drp1 self-assembly. Moreover, we now show that, Mff-decorated ESL templates were efficiently remodeled by wild-type Drp1 to form extended tubular structures. We also assessed the roles of various mitochondrial lipids, including the negatively charged cardiolipin and the conical lipid PE. Cardiolipin synergizes with Mff to further enhance Drp1 self-assembly, while the membrane flexibility and fluidity imparted by PE enhances membrane tubulation but does not further augment Mff-induced stimulation.
To assess ultrastructural changes in membrane morphologies, EM analyses were required. Drp1 GTPase activity was elevated through clustering and assembly of filamentous polymers that did not reshape the liposomes to any great extent2. However, membrane deformation was observed when the more mitochondria-like SL/PE/CL template was used. Interestingly, the enzyme activity was not enhanced. Therefore, the EM studies were essential in identifying key differences that would otherwise be missed using the functional assay.
While this technique is powerful for exploring the function and interaction of soluble proteins and soluble protein domains, these lipid scaffolds cannot account for the role of transmembrane domains. This is an important consideration because the transmembrane domain can effect dynamic protein processes such as self-assembly27 and lateral diffusion28-30 in lipid bilayers. If these factors are critical for evaluating protein interactions at the membrane surface, then traditional lipid reconstitution experiments with detergent would be favored. Alternative tethers may also be explored to control the recruitment and mobility of the membrane anchored proteins.
In addition to using His-tagged proteins with NTA(Ni2+) lipid anchors, other tethers such as biotin-conjugated31 or reactive group-conjugated lipids can be utilized. These covalent modifications would more stably trap proteins at the lipid surface, but mobility and exchange of these factors would likely be diminished. As such, the tether should be carefully considered in the context of the protein complexes being studied. When considering the use of this method, the mode of tethering proteins to lipid templates have the potential to influence certain assays. For instance, the His-tag tethering to NTA(Ni2+) method may be more appropriate for in situ assays rather than separation experiments, especially in the case of transient protein-protein interactions. This is clearly demonstrated in Figure 3 by the discrepancy between the in situ negative stain electron microscopy and the sedimentation assay.
In the future, a combination of two or more of these anchor lipids with distinct target headgroups could be implemented to allow for recruitment of multiple proteins to a scaffold template without competition for a single lipid tether. Moreover, the relative abundance of each component can be managed by altering the lipid composition. Additional lipid cofactors, such as phosphoinositides, cardiolipin, and phosphatidylserine, can easily be introduced in these templates to assess the isolated impact of a variety of factors.
Overall, these lipid scaffolds represent a novel platform for investigating complex protein interactions near lipid membranes. These templates are easily generated and are simple to tailor to a range of diverse applications, including enzymatic assays, electron microscopy, or fluorescence imaging. In addition, the lipid composition can be formulated to resemble an organelle or membrane microdomain of interest to better recapitulate protein function at these specific regions. Using these techniques, biochemists can probe the complex interactions of membrane bound and membrane associated proteins with their partners and their environment.

Studying membrane-proximal protein-protein interactions is a challenging endeavor due to difficulty in recapitulating the native environment of the integral membrane proteins involved1. This is due to the necessity of detergent solubilization and the inconsistent orientation of proteins in proteoliposomes. In order to avoid these issues, we have employed a strategy whereby soluble domains of integral membrane proteins are expressed as His-tag fusion proteins, and these soluble fragments are anchored to scaffold liposomes via interactions with NTA(Ni2+) headgroups at the lipid surface. Using these scaffolds, lipid-proximal protein interactions can be investigated over a range of lipid and protein compositions.
We have effectively applied this method to investigate the critical protein-protein interactions that govern assembly of the mitochondrial fission complex and examine lipid interactions that modulate this process2. During mitochondrial fission, a conserved membrane remodeling protein, called Dynamin-related protein 1 (Drp1)3, is recruited to the surface of the Outer Mitochondrial Membrane (OMM) in response to cellular signals that regulate energy homeostasis, apoptotic signaling, and several other integral mitochondrial processes. This large, cytosolic GTPase is recruited to the surface of mitochondria through interactions with integral OMM proteins4-8. The role of one such protein, Mitochondrial Fission Factor (Mff), has been difficult to elucidate due to an apparent weak interaction with Drp1 in vitro. Nevertheless, genetic studies have clearly demonstrated that Mff is essential for successful mitochondrial fission7,8. The method described in this manuscript was able to overcome previous shortcomings by introducing simultaneous lipid interactions that promote Drp1-Mff interactions. Overall, this novel assay revealed fundamental interactions guiding assembly of the mitochondrial fission complex and provided a new stage for ongoing structural and functional studies of this essential molecular machine.
To date, examination of interactions between Drp1 and Mff have been complicated by the inherent flexibility of Mff9, the heterogeneity of Drp1 polymers2,10, and the difficulty in purifying and reconstituting full-length Mff with an intact transmembrane domain11. We addressed these challenges by using NTA(Ni2+) scaffold liposomes to reconstitute His-tagged Mff lacking its transmembrane domain(Mff&#916;TM-His6). This strategy was advantageous because Mff&#916;TM was extremely soluble when over-expressed in E. coli, and this isolated protein was easily reconstituted on scaffold liposomes. When tethered to these lipid templates, Mff assumed an identical, outward facing orientation on the surface of the membrane. In addition to these advantages, mitochondrial lipids, such as cardiolipin, were added to stabilize Mff folding and association with the membrane11. Cardiolipin also interacts with the variable domain of Drp12,12 which may stabilize this disordered region and facilitate assembly of the fission machinery.
This robust method is widely applicable for future studies that seek to evaluate membrane-proximal protein interactions. Through the use of additional tethering/affinity interactions, the sophistication of these membrane reconstitution studies can be enhanced to mimic additional complexity found at the surface of membranes within cells. At the same time, lipid compositions can be modified to more accurately mimic the native environments of these macromolecular complexes. In summary, this method provides a means to examine the relative contributions of proteins and lipids in shaping membrane morphologies to during critical cellular processes.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylcholine (DOPC)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850375</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylethanolamine (DOPE)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:287px;">DGS-NTA(Ni2+)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">790404</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">KOH</td>
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			<td style="width:136px;">P250</td>
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			<td height="21" style="height:21px;width:287px;">Magnesium Chloride</td>
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			<td height="21" style="height:21px;width:287px;">4-20% SDS-PAGE Gel</td>
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			<td style="width:136px;">456-1096</td>
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			<td style="width:136px;">161-0747</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">HCL</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Malachite Green Carbinol</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">229105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ammonium Molybdate Tetrahydrate</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">A7302</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Laboratory Film</td>
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			<td height="21" style="height:21px;width:287px;">Uranyl Acetate</td>
			<td style="width:229px;">Polysciences</td>
			<td style="width:136px;">21447</td>
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			<td style="width:136px;"></td>
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			<td height="21" style="height:21px;width:287px;">Optima MAX</td>
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			<td height="21" style="height:21px;width:287px;">TLA-55 Rotor</td>
			<td style="width:229px;">Beckman</td>
			<td style="width:136px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Refrigerated CentriVap Concentrator</td>
			<td style="width:229px;">Labconico</td>
			<td style="width:136px;"></td>
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			<td height="21" style="height:21px;width:287px;">Mastercycler Pro Thermocycler</td>
			<td style="width:229px;">Eppendorf</td>
			<td style="width:136px;"></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">VersaMax Microplate reader</td>
			<td style="width:229px;">Molecular Devices</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

While the interaction between Drp1 and Mff has been demonstrated to be important for mitochondrial fission, this interaction has been difficult to recapitulate in vitro. Our goal was to better emulate the cellular environment wherein Drp1 and Mff interact. To this end, liposomes containing limiting concentrations of NTA(Ni2+) headgroups were prepared by rehydrating a lipid film as described above. The lipid solution initially consists of unilamellar and multilamellar vesicles of heterogeneous diameters as evidenced by the opacity of the solution (Figure 1a). This opacity is reduced by freeze-thawing (Figure 1b), which reduces the prevalence of multilamellar vesicles. The liposome diameters are further homogenized by extrusion through a polycarbonate filter, which results in a clear solution (Figure 1c).
In previous studies, we found that Drp1 was able to assemble on Mff-decorated scaffold liposomes, and membrane tubulation was observed when flexible membranes were employed2. Building on these findings, we utilized a new template composed of PC, PE, Ni, and CL (called Enriched Scaffold Liposomes or ESL) to promote ordered assembly of a polymeric Drp1-Mff complex capable of inducing membrane deformation. Specifically, increased NTA(Ni2+) and cardiolipin lipids were utilized (10 mol% and 15 mol% respectively) for this application. Then, GFP or Mff was tethered to ESL templates in the presence and absence of Drp1 (Figure 2), and the ability of Drp1 to remodel membranes was qualitatively assessed. In the absence of Drp1, neither Mff nor GFP resulted in membrane deformation (Figure&#160;3a,&#160;b), and similarly in the case of GFP-decorated ESL, only featureless liposomes were observed (Figure 3c). However, when Drp1 was added to Mff-decorated ESL templates, remodeling of the liposomes was evident (Figure 3d).
While macromolecular complex formation clearly demonstrates an interaction between Drp1 and Mff, this qualitative analysis alone is incapable of determining the functional effects of such an interaction. Therefore, we utilized a malachite green phosphate generation assay18 to assess alterations in the catalytic activity of Drp1 in response to interaction with Mff. As described previously2, we initially utilized a simple scaffold liposome (SL; 3.3 mol% DGS-NTA(Ni2+), 96.7 mol% DOPC) to investigate the effect of Mff alone on Drp1 structure and function. Nonspecific interaction of Drp1 with NTA(Ni2+) has previously been described20, so SL was initially designed to contain low concentrations of NTA(Ni2+) to avoid nonspecific activity stimulation of Drp1. With the larger amounts of NTA(Ni2+) in ESL, the use of His-tagged GFP as a control was found to be critical to shield the Ni2+ and prevent non-specific Drp1 interactions. After decoration of SL liposomes by Mff or GFP (as illustrated in Figure 2), the extent of self-assembly can be assessed by measuring the GTPase activity of Drp1. In the absence of liposomes, Drp1 has a relatively low basal GTPase activity, which is slightly enhanced by addition of SL. Decoration of these scaffold liposomes with Mff enhanced GTPase activity (Figure 4a, 1.8 fold). Conversely, when the exposed NTA(Ni2+) headgroups were blocked with His-tagged GFP, this augmented GTPase activity was ablated. We also tested the role of Fis1, an OMM protein that has been suggested to have a role in mitochondrial fission21,22, though this has been challenged in recent studies7,23. Tethering of Fis1 lacking its transmembrane domain to SL also failed to elicit a stimulation of Drp1&#39;s GTPase activity (Figure 4a).
We then utilized a slightly more complex lipid scaffold containing a small amount of cardiolipin (SL/CL: SL with 10 mol% cardiolipin replacing DOPC) to determine the role of this mitochondrial lipid in the interaction of Drp1 and Mff. This moderate concentration of cardiolipin was specifically chosen to limit the stimulation of Drp1 by cardiolipin as described previously10. Similar to SL, addition of SL/CL to Drp1 resulted in a slight stimulation of GTPase activity that was reversed by tethering His-tagged Fis1 or GFP to the liposomes. A synergy between Mff and cardiolipin was observed as the GTPase activity of Drp1 was stimulated 2.6&#160;fold when it was incubated with Mff-decorated SL/CL (Figure 4b).
Membrane fluidity and the ability of Drp1 to remodel lipid bilayers have been proposed to enhance its GTPase activity. Therefore, we sought to examine the effect of membrane fluidity/flexibility using a flexible scaffold liposome. This was achieved by replacing 35 mol% of DOPC in SL/CL with DOPE (SL/PE/CL), which has previously been shown to allow for Drp1-mediated membrane remodeling10. Addition of undecorated SL/PE/CL scaffold liposomes to Drp1 slightly enhances Drp1 GTPase activity, and decoration of these liposomes with GFP eliminates this effect. When SL/PE/CL templates were decorated with Mff, Drp1 activity was enhanced (Figure 4c, 2.4&#160;fold). As we have previously shown, the ability of Drp1 to remodel liposomes into lipid tubules was enhanced by the addition of PE to the scaffold liposomes. Interestingly, this improved tubulation leading to the formation of a helical Drp1 polymer did not result in any greater stimulation when compared to liposomes that Drp1 was unable to remodel2.
Using these adaptable lipid templates, Mff and Drp1 were found to interact in a more native environment in vitro. This technique has enabled us to control the relative abundance of Drp1, Mff (through NTA(Ni2+) concentration), and specific lipids (cardiolipin and PE specifically) that appeared to regulate the assembly of this macromolecular complex. As we have demonstrated, this method can be utilized to visualize the membrane remodeling of Mff-recruited Drp1 by electron microscopy, and to determine the effects of Drp1 assembly on its catalytic activity using GTPase activity assay.
<img alt="Figure 1" src="/files/ftp_upload/54971/54971fig1.jpg" /> 
Figure 1: Lipid Preparation Schematic. (a) Upon resuspension, liposomes of diverse sizes form and consist of unilamellar and multilamellar vesicles, which results in an opaque solution (inset). (b) Freeze-thawing the solution results in a more unilamellar population of liposomes, which are still heterogeneous in diameter. Freeze-thawing clarifies the solution (inset). (c) Extrusion of the lipid solution homogenizes the liposome diameter (1.0 &#181;m in this example), and results in a clear solution (inset). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54971/54971fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54971/54971fig2.jpg" /> 
Figure 2: Methods to Assess Protein assembly. A schematic depicting partner protein assembly on scaffold liposomes is presented. His-tagged partner proteins or GFP are incubated with scaffold liposomes, and then Drp1 is incubated with decorated or undecorated liposomes. These Drp1-preassembled liposomes can then be analyzed by structural methods (electron microscopy) and functional assays (GTPase assay). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54971/54971fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54971/54971fig3.jpg" /> 
Figure 3: Structural Assessment of Drp1 Recruitment. Negative stain transmission micrographs of GFP or Mff decorated liposomes alone (A,&#160;B, respectively) or incubated with Drp1 (C,&#160;D, respectively). Scale bar = 100 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54971/54971fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54971/54971fig4.jpg" /> 
Figure 4: Scaffold Liposome Enzymatic Assay. (A - C) The generation of phosphate over time was measured (inset), and the kcat was determined. This method was applied to SL-tethered proteins (A), SL/CL-tethered proteins (B), or SL/PE/CL-tethered proteins (C). #: p &#60;0.05, *: p &#60;0.0001, **: p &#60;0.000001 as determined by unpaired student&#39;s T-test. All error bars represent standard deviation from 3 independent samples. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54971/54971fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

using-scaffold-liposomes-to-reconstitute-lipid-proximal-protein

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.7K Views.  Case Western Reserve University School of Medicine. This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment. 

Video: Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...