This work was supported by the Deutsche Forschungsgemeinschaft under grants SFB 858/B04, EXC 1003, SFB 1348/A04, and SFB 1348/A11.

NOTE: Buffers should be filtered using a 0.22-&#181;m filter and degassed by a vacuum for 1 h.
1. Lipid Vesicle Preparation
<ol>
	<li>Use 2-mL glass tubes. Dissolve each lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesterol (Chol), in a mixture of chloroform/methanol (50:50 v/v) to prepare a clear 5 mM lipid solution. Dissolve 1,2-dioleoyl-sn-glycero-3-phospho-(1&#39;-myo-inositol-4&#39;,5&#39;-bisphosphate) (PI(4,5)P2) in a mixture of chloroform/methanol/water (20:9:1 v/v).</li>
	<li>Combine the dissolved lipids in the desired molar ratio of POPC/POPS (80/20), POPC/PI(4,5)P2 (95/5), POPC/POPS/Chol (60/20/20), POPC/POPS/PI(4,5)P2/Chol (60/17/3/20), and POPC/DOPC/POPS/PI(4,5)P2/Chol (37/20/20/3/20) (Table 1) in 10-mL glass tubes. 
	NOTE: The final amount of total lipid is 500 &#181;g.</li>
	<li>Evaporate the organic solvents using a dry stream of nitrogen. Leave the lipid mixture on a high vacuum (lyophilization) system for 3 h to remove any residual traces of the solvents. 
	NOTE: This results in a dry clear film.</li>
	<li>Resuspend the lipid film in 1 mL of citrate buffer (10 mM trisodium-citrate, 150 mM NaCl, pH 4.6). Incubate the lipid suspension at 60 &#176;C (this temperature is around 10 &#176;C above the phase transition temperature of the highest melting lipid in the mixture) for 30 min in a water bath, and vortex it vigorously every 5 min. 
	NOTE: This results in the formation of large, multilamellar vesicles (MLVs). Keep the suspension above the transition temperature.</li>
	<li>Preheat the extruder (at 60 &#176;C in this case) equipped with a 50-nm diameter pore-size polycarbonate membrane above the transition temperature (which is 40 - 50 &#176;C here) for 30 min.</li>
	<li>Load the MLV suspension into the preheated extruder and gently pass the mixture 31x through the polycarbonate membrane to form small unilamellar vesicles (SUVs)14. Keep the temperature above the transition temperature.</li>
	<li>Transfer the SUV suspension to a 2&#160;mL plastic reaction vessel and add the citrate buffer (see step 1.4) to bring the final volume to 2 mL. 
	NOTE: This will yield a final lipid concentration of 250 &#181;g/mL.</li>
</ol>
2. Handling the Quartz Sensors
NOTE: Always handle the quartz sensors with a tweezer.
<ol>
	<li>Incubate four sensors inserted in a polytetrafluoroethylene holder in a 2% SDS solution for &#8805; 30 min. Wash them extensively with ultra-pure water to completely remove the SDS and let them dry using a stream of dry argon or nitrogen.</li>
	<li>Use a plasma-cleaning system to completely remove any contaminants. Insert the dry sensors in the plasma-cleaning chamber, evacuate the chamber, and flush it 3x with oxygen. Turn the plasma cleaner on. Use the following process parameters: 1 x 10-4 Torr pressure, high radio frequency (RF) power, and 10 min of process time. Turn off the machine and take out the sensors.</li>
</ol>
3. Microbalance Operation
NOTE: A microbalance system with four temperature-controlled flow chambers in a parallel configuration, connected to a peristaltic pump and set to a flow rate of 80 &#181;L/min, was used. In the open flow mode, the buffer was pumped from the feeder reservoir into the receiving tank. In the loop mode, the receiving tank was connected with the feeder reservoir to generate a closed loop. The temperature was set to 20 &#176;C.
<ol>
	<li>Carefully dock the plasma-cleaned sensors into the 4 flow chambers, using tweezers. Avoid any pressure on or torsion of the chambers and the tubes that might cause leaking.</li>
	<li>Flush the system with citrate buffer (10 mM trisodium-citrate, 150 mM NaCl, pH 4.6) in the open-flow mode for 10 min. 
	NOTE: This requires exactly 3.2 mL of buffer, but it is advisable to use an excess of buffer (10 mL).</li>
	<li>Launch the program. Start recording any changes in the frequency and dissipation of the first fundamental tone (n = 1st) and overtones (n = 3rd - 13th) using the software, until the frequency and dissipation baselines are stable (this will take around 40 - 60 min). 
	NOTE: Frequency noise levels (peak-to-peak) should be lower than 0.5 Hz and, for the dissipation, lower than 0.1&#183;10-6, with a maximal drift (in aqueous solution) of 1 Hz/h in frequency and 0.3&#183;10-6/h in dissipation.</li>
	<li>When the baselines are stable, apply the SUV suspension in citrate buffer (2 mL in a small tube). Using a reaction vessel, remove 1.5 mL of the dead volume. Then close the system in the loop-flow mode. Record the frequency/dissipation shift for another 10 minutes. 
	NOTE: During this time, the vesicles will spread onto the SiO2 surface and fuse to form a continuous bilayer15,16&#160;(step 2 in Figures 1, Figure&#160;2, and Figure&#160;3). The SUVs adsorption on the surface of the sensor is two-phasic and has a typical frequency minimum and maximum in the dissipation. A stable new frequency/dissipation baseline with a characteristic frequency shift (depending on the lipid composition) from 26 - 29 Hz (see Table 1) indicates a continuous bilayer on the surface.</li>
	<li>When the SLB is stable (see step 3.4), equilibrate the system with the running buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) at the required Ca2+ concentrations (ranging from 50 &#181;M up to 1 mM CaCl2, depending on the experiment) in an open flow mode for 40 min.</li>
	<li>Add the protein (here, AnxA2) to the running buffer containing Ca2+ (see step 3.5). Perform the application of the protein in a loop-flow mode until an equilibrium steady state is reached (step 3 in Figure&#160;1, Figure 2, and Figure 3). 
	NOTE: The protein concentration may range from 1 to 400 nM. Protein adsorption results in a concentration-dependent frequency shift reflecting the mass (protein) adsorption.</li>
	<li>Dissociate the bound protein by chelating Ca2+ ions with 5 mM EGTA in the running buffer in open flow mode (step 4 in Figures 1 and Figure&#160;2). 
	NOTE: A recovery of the frequency and dissipation to the SLB baseline indicates a total reversibility of protein binding. Association-dissociation cycles can be repeated to compare different concentrations or proteins.</li>
</ol>
4. Microbalance Cleaning
NOTE: Perform a minimal cleaning procedure after each measurement.
<ol>
	<li>Regenerate the microbalance system with 50 mL of ddH2O in a continuous open flow mode, remove the tubes from the water container, and let the system run dry.</li>
	<li>Carefully remove the crystal sensor and clean it with the 2% SDS solution using the polytetrafluoroethylene holder (see step 2.1).</li>
	<li>Dry the visible parts of the flow module interior where the sensor was placed. 
	NOTE: Perform an intensive cleaning procedure after a series of 10 measurements.</li>
	<li>Clean the system with a 2% SDS solution (50 mL) at 40 &#176;C (setup in the software) in a continuous open flow (tube) mode, using a flow rate of 20 &#181;L/min for extended contact with the detergent.</li>
	<li>Clean with 250 mL of ddH2O in continuous open flow mode at a flow rate of 160 &#181;L/min. 
	NOTE: After 4 months, carry out an extensive cleaning procedure according to the manufacturer&#39;s manual.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+curvature+and+its+generation+by+BAR+proteins.">Membrane curvature and its generation by BAR proteins.</a> Trends in Biochemical Science. 37, 526-533 (2012).</li><li>Rescher, U., Ruhe, D., Ludwig, C., Zobiack, N., Gerke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+2+is+a+phosphatidylinositol+(4,5)-bisphosphate+binding+protein+recruited+to+actin+assembly+sites+at+cellular+membranes.">Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes.</a> Journal of Cell Science. 117, 3473-3480 (2004).</li><li>Gerke, V., Moss, S. 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The authors have nothing to disclose.

To answer questions concerning the structure-function relationship of cellular membranes both in a quantitative and qualitative manner, cell biology profits immensely from the use of biophysical approaches based on well-established and widely used techniques, including atomic force microscopy (AFM), surface plasmon resonance (SPR), and the QCM-D technique employed here. We showed in previous studies that annexin proteins bind in a Ca2+-dependent manner to the immobilized membrane with high affinity. We use frequency and dissipation shifts of the 7th overtone (&#916;f7) because this represents the best compromise of detection sensitivity and oscillation stability.
This technique also permits a quantitative description of the membrane-protein interaction. AnxA2 binding to the membrane is characterized by positive cooperativity that is mediated by the conserved annexin core domain and depends on the presence of cholesterol. The quantitative data obtained for AnxA2 and AnxA8 are reported in detail elsewhere6,8.
There are many critical steps in this protocol. Use the liposomes immediately; otherwise, small vesicles might fuse into bigger vesicles with less surface tension, leading to the inhibition of the lipid bilayer formation. Maintain a constant temperature during the measurements. Each small deviation in temperature results in a not negligible frequency and dissipation shift. Avoid air bubbles; otherwise, the system is unstable and will not establish a baseline.
The Sauerbrey equation allows the direct conversion of the observed frequency changes into changes in mass and is, therefore, widely used. However, the assumption of a linear correlation between the change in resonance frequency and the added mass only holds true for components forming a rigid and uniform film on the sensor surface. The Sauerbrey equation cannot be used for viscoelastic adsorbents such as water-rich protein films, lipid layers with incorporated water, or even adsorbed cells. Here, more complex mathematical models are required. Therefore, it is extremely important to simultaneously monitor the changes in frequency and dissipation. To detect structural changes during the measurement, &#916;D versus &#916;f ratios can be plotted, with a straight line indicating no conformational changes.
A major advantage of this technique is the possibility to use a very broad range of materials as substrates. Moreover, it is a reliable and direct method to study a broad range of macromolecular interactions, as the proper formation of the surface-coating films (such as SLB), as well as further protein-lipid interactions, can be monitored online.
This protocol can be applied to other membrane-interacting proteins, for example, BAR domain proteins19, the ERM (ezrin, radixin, and moesin) protein family that has an important role in membrane-cytoskeleton linkage20,21,22, or proteins containing C2 or PH domains. Moreover, a wide range of applications of this technique to study biological materials has been successfully published, thus establishing QCM as a well-suited experimental platform to study the interactions of more complex macromolecular assemblies or even cells23,24.

Cellular membranes are highly dynamic and complex structures. The compound mixture of membrane lipids, together with peripherally and/or integrally associated membrane proteins, assemble to form microdomains. The coordinated tempo-spatial organization of these membrane microdomains is involved in key physiological processes1. Membrane microdomain dynamics are driven by the interplay of membrane lipids, as well as by the ability of peripheral membrane proteins to recognize and interact with lipids enriched in the microdomains. The recruitment of the proteins to the specific lipids is often achieved via lipid-recognition modules, such as the pleckstrin homology (PH) or the C2 domains2,3. Biophysical analytical methods using model membranes are key to understanding the fundamental principles governing these processes at the molecular level.
Annexins, a large multigene-family, are well-known for their ability to bind to negatively charged membrane lipids, predominantly phosphatidylserine (PS), in a Ca2+-controlled manner2. The second hallmark of the annexin family is the presence of a conserved structural segment, the annexin repeat, that is present four or eight times and harbors the Ca2+- and phospholipid-binding sites4. The Ca2+-dependent lipid interaction places the annexins in a perfect position to sense and convey Ca2+-mediated signaling to target membranes. Consistently, annexins are able to induce the formation of microdomains enriched in cholesterol, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and PS, both in cellular and/or artificial membrane systems5. This protocol describes an approach to analyze the annexin-membrane interaction using a Quartz Crystal Microbalance Dissipation (QCM-D)6,7,8.
The basic component in this microbalance is an oscillating crystal that serves as the sensor surface. The adsorption and/or binding of molecules to the sensor surface decreases the resonance frequency (f) proportional to the increase in mass. If the surface is evenly coated with a film, the binding of additional substances may interfere with the structural integrity of this layer, and such changes in viscoelasticity (the energy dissipation factor D) can be additionally monitored. This is a widespread technique to study the interaction of proteins with lipid bilayers. In this approach, lipid vesicles are absorbed onto the appropriately coated sensor surface. Lipid bilayer formation is favorable on silica-based materials9,10, as vesicles often do not rupture on other hydrophilic surfaces, such as gold11&#160;oxidized after UV-ozone exposure, TiO212, or Al2O313. The rupture of the coalescing vesicles releases the aqueous phase, leading to characteristic changes in mass and dissipation. The generation of solid-supported bilayers (SLB) by vesicle fusion is simple and robust and can be used to generate complex models that mimic cellular membranes.
Dissipative quartz crystal microbalance is a label-free and sensitive technique. A major advantage is the possibility to coat any material that generates a sufficiently thin film onto the surface, thus providing a broad range of applications in diverse research areas. The protein-membrane interaction is observed in real-time, and the results can be analyzed directly. The same sensor surface can be used in subsequent measurements (after performing a minimal cleaning as described in this protocol), thus allowing for accurate internal controls and a comparability between analytes.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calciumchloride</td><td>Merck</td><td>017-013-00-2</td><td>99%</td></tr><tr><td>Chloroform</td><td>Roth</td><td>4432.1</td><td>99%</td></tr><tr><td>DOPC</td><td>Avanti</td><td>850375P</td><td></td></tr><tr><td>EGTA</td><td>PanReac AppliChem</td><td>A0878</td><td>99%</td></tr><tr><td>HEPES</td><td>PanReac AppliChem</td><td>A1069</td><td></td></tr><tr><td>Methanol</td><td>PanReac AppliChem</td><td>A3493</td><td></td></tr><tr><td>PiP&lt;sub&gt;2&lt;/sub&gt;</td><td>Avanti&amp;nbsp;</td><td>850155P</td><td></td></tr><tr><td>POPC</td><td>Avanti&amp;nbsp;</td><td>850457P</td><td></td></tr><tr><td>POPS</td><td>Avanti&amp;nbsp;</td><td>840034P</td><td></td></tr><tr><td>Sodiumchloride</td><td>PanReac AppliChem</td><td>A1149</td><td></td></tr><tr><td>SDS</td><td>Roth</td><td>183</td><td></td></tr><tr><td>Trisodium citrate</td><td>PanReac AppliChem</td><td>A3901</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Extruder Liposofast</td><td>Avestin</td><td></td><td></td></tr><tr><td>Qsense E4 Analyzer</td><td>Qsense</td><td></td><td></td></tr><tr><td>QSense Dfind</td><td>Qsense</td><td></td><td></td></tr><tr><td>Pump IPC 4</td><td>Ismatec</td><td>ISM 930</td><td></td></tr><tr><td>QSX 303 SiO&lt;sub&gt;2&lt;/sub&gt; Silicon dioxide 50nm</td><td>Qsense</td><td>QSX 303</td><td></td></tr><tr><td>PC Membranes 0.05&amp;mu;m</td><td>Avanti polar lipids</td><td>610003</td><td></td></tr><tr><td>OriginPro</td><td>OriginLab Corporation</td><td></td><td>Version 8 and 9</td></tr></tbody></table>

dissipative microgravimetry to study the binding dynamics of the phospholipid binding protein annexin a2 to solid-supported lipid bilayers using a quartz resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

The decrease in the resonant frequency (&#916;f) correlates in a linear manner with the adsorbed mass (&#916;m), as defined by the Sauerbrey equation.17
<img alt="Equation 1" src="/files/ftp_upload/58224/58224eq1.jpg" />
Here, f is the resonant frequency, Cf is a constant that depends on the geometrical and physical characteristics of the given quartz and the resonant frequency, and A is the sensor surface area.
In most applications, the adsorbed layer is not completely rigid but viscoelastic. The resulting dampening of the quartz sensor oscillation is referred to as dissipation (D). The monitored dissipation changes (&#916;D) correlate with the viscoelastic properties of the bound mass18&#160;and are defined as follows8.
<img alt="Equation 2" src="/files/ftp_upload/58224/58224eq2.jpg" />
Here, Edissipated is the energy lost during one oscillation period, and Estored is the total energy of the freely oscillating sensor.
To analyze and quantify the binding parameters, frequency isotherms are derived by plotting the equilibrium frequency shifts (&#916;&#916;fe) against the protein concentrations. &#916;&#916;fe is defined as
<img alt="Equation 3" src="/files/ftp_upload/58224/58224eq3.jpg" />
Here, &#916;ft1&#160;represents the beginning of the protein adsorption and &#916;ft2&#160;the equilibrium state. Nonlinear curve fitting can be performed by using a Hill expansion of the Langmuir equation as follows6,8.
<img alt="Equation 4" src="/files/ftp_upload/58224/58224eq4.jpg" />
Here, &#916;&#916;fmax is the &#916;&#916;fe of the protein concentration resulting in maximum (saturating) binding, Kd is the apparent dissociation constant for the protein/membrane complex, and n is the Hill coefficient.
The Hill coefficient (n) describes the cooperativity of binding. For n = 1, the Hill adsorption model is a simple Langmuir isotherm (the equal binding sites and all molecules bind independently of each other to the lipid bilayer). If n &#8800; 1, a bound ligand changes the membrane binding affinity for other ligands, either increasing (n &#62; 1, positive cooperativity) or decreasing (n &#60; 1, negative cooperativity) the affinity.
Figure 1 shows a schematic of the experimental workflow used in our laboratory to measure the shifts in resonance and frequency during Ca2+-dependent binding and the release of AnxA2 to the lipid bilayer in the liquid phase. An exemplary recording is shown in Figure 2. Figure 2A shows the recording of the frequency curve and Figure 2B shows the dissipation shifts. The prominent drop in frequency upon the addition of the liposomes (Figure 2A [step 1]) indicates their adsorption. Because the buffer-filled vesicles are not rigid, but viscoelastic, the dissipation increases (Figure 2B [step 1]). Subsequently, the coalescing vesicles rupture. The concomitant release of the buffer inside the vesicles decreases the adsorbed mass until a stable plateau is reached (Figure 2A [step 2]). Of note, the addition of vesicles results in a high dissipation shift, while the shift in response to the bilayer is much smaller due to the rigid homogenous nature of the SLB (Figure 2B [step 2]). Step 3 in Figure&#160;2A and 2B records the binding of AnxA2 to the lipids, which adds mass, as seen by the clear frequency shift, but does not interfere with the bilayer structure, as indicated by the only small change in dissipation. When Ca2+ is removed by the chelating agent EGTA (Figure&#160;1 and Figure&#160;2 [step 4]), AnxA2 dissociates from the lipid film. The frequency, as well as the dissipation recordings, shift to the levels seen with the bilayer only (compare steps 2 and 4 in Figure&#160;2A and 2B), indicating that AnxA2 binding is totally dependent on Ca2+ and that the lipid film remains intact.
AnxA2, as do most of the annexins, depends on negatively charged lipids such as PS. This is clearly seen when POPS is absent in the lipid bilayer (Figure 3). Figure 3A shows the recording of the frequency curve and Figure 3B shows the dissipation shifts. Note that the frequency shifts to a stable baseline at -25 Hz, yet the dissipation is not altered (Figure 3B [step 2]), indicative of a proper bilayer formation. However, no changes in frequency (Figure 3A) or dissipation (Figure 3B) are observed after the addition of AnxA2 in the presence of Ca2+ (Figure&#160;3A and 3B [step 3]) or EGTA (Figure&#160;3A and 3B [step 4]), as AnxA2 cannot interact with the lipid film.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58224/58224fig1.jpg" /> 
Figure 1:&#160;Graphical model of the experimental workflow. This workflow illustrates the vesicle absorption to the hydrophilic sensor surface (step 1), the vesicle fusion/rupture leading to the SLB formation (step 2), and the Ca2+-dependent adsorption (step 3) and EGTA-dependent desorption of AnxA2 (step 4). <a href="https://www.jove.com/files/ftp_upload/58224/58224fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58224/58224fig2.jpg" /> 
Figure 2: Exemplary recording. These panels show (A) the time-dependent monitoring of the 7th overtone resonance frequency and (B) the dissipation shifts of the quartz sensors during measurement. The application of the liposomes causes a rapid drop in the frequency baseline, whereas the dissipation baseline increases (step 1). The stabilization of the baselines indicates the formation of the bilayer (step 2). The AnxA2 (200 nM) adsorption (in the presence of Ca2+) onto the POPS-containing lipid bilayer adds mass without significantly changing the dissipation, indicating that the lipid film is not perturbed (step 3). The recovery of the frequency baseline upon Ca2+ chelation with EGTA indicates the total desorption of the protein (step 4). <a href="https://www.jove.com/files/ftp_upload/58224/58224fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58224/58224fig3.jpg" /> 
Figure 3:&#160;Negative control experiment, demonstrating that AnxA2 does not bind to SLBs in the absence of POPS. These panels show the addition of liposomes and the SLB formation (steps 1 and 2). No changes in (A) frequency or (B) dissipation are apparent after the addition of AnxA2 (step 3; 200 nM, in the presence of Ca2+) or EGTA (step 4). <a href="https://www.jove.com/files/ftp_upload/58224/58224fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Composition</td>
			<td>&#916;&#916;F/Hz after formation of SLBs</td>
			<td>&#916;&#916;D*10-6 after formation of SLB</td>
		</tr>
		<tr>
			<td>POPC/POPS 
			(80 : 20)</td>
			<td>26.3 &#177; 0.2</td>
			<td>0.26 &#177; 0.03</td>
		</tr>
		<tr>
			<td>POPC/PI(4,5)P2 
			(95 : 5)</td>
			<td>26.5 &#177; 0.5</td>
			<td>0.31 &#177; 0.02</td>
		</tr>
		<tr>
			<td>POPC/POPS/Chol 
			(60 : 20 : 20)</td>
			<td>29.2 &#177; 0.2</td>
			<td>0.45 &#177; 0.09</td>
		</tr>
		<tr>
			<td>POPC/POPS/ PI(4,5)P2/Chol 
			(60 : 17 : 3 : 20)</td>
			<td>29.6 &#177; 0.6</td>
			<td>0.43 &#177; 0.10</td>
		</tr>
		<tr>
			<td>POPC/DOPC/POPS/ PI(4,5)P2/Chol 
			(37 : 20 : 20 : 3 : 20)</td>
			<td>29.4 &#177; 0.4</td>
			<td>0.39 &#177; 0.14</td>
		</tr>
	</tbody>
</table>
Table 1: Lipid composition and formation data of the SLB7.

Watch this Scientific Journal Video about Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Reso

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

dissipative-microgravimetry-to-study-binding-dynamics-phospholipid

Research

JoVE Journal

Biochemistry

7.0K Views.  University of Münster. Deze methode kan helpen bij het bestuderen van richtingen van eiwitten met lipidemembranen. In ons geval, gebruik het om de calciumafhankelijke binding van annexins te analyseren aan negatief geladen fosfolipiden. De belangrijkste voordelen van deze techniek zijn dat het label vrij, gevoelig, kwantitatief, en dat interacties kunnen worden waargenomen in real time.Aldus waardoor directe analyse van de werkelijke resultaten mogelijk is. Deze techniek kan ook worden toegepast op andere sy...

Video: Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Chemistry

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Bioengineering

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of the data and determines which properties of the film can be quantified. The quartz crystal microbalance offers a uniquely sensitive platform for measuring fine changes in mass and/or mechanical properties of an applied film by observing the changes in mechanical resonance of a quartz crystal oscillating at high frequency. The advantages of this approach include its experimental versatility, ability to study changes in properties over a wide range of experimental time lengths, and the use of small sample sizes. We demonstrate that, based on the thickness and shear modulus of the layer deposited on the sensor, we can acquire different information from the material. Here, this concept is specifically exploited to display experimental parameters resulting in mass and viscoelastic calculations of adsorbed collagen on gold and polyelectrolyte complexes during swelling as a function of salt concentration.

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of ...

Epitaxial Nanostructured &alpha;-Quartz Films on Silicon: From the Material to New Devices

In this work, we show a detailed engineering route of the first piezoelectric nanostructured epitaxial quartz-based microcantilever. We will explain all the steps in the process starting from the material to the device fabrication. The epitaxial growth of &#945;-quartz film on SOI (100) substrate starts with the preparation of a strontium doped silica sol-gel and continues with the deposition of this gel into the SOI substrate in a thin film form using the dip-coating technique under atmospheric conditions at room temperature. Before crystallization of the gel film, nanostructuration is performed onto the film surface by nanoimprint lithography (NIL). Epitaxial film growth is reached at 1000 &#176;C, inducing a perfect crystallization of the patterned gel film. Fabrication of quartz crystal cantilever devices is a four-step process based on microfabrication techniques. The process starts with shaping the quartz surface, and then metal deposition for electrodes follows it. After removing the silicone, the cantilever is released from SOI substrate eliminating SiO2 between silicon and quartz. The device performance is analyzed by non-contact laser vibrometer (LDV) and atomic force microscopy (AFM). Among the different cantilever&#8217;s dimensions included in the fabricated chip, the nanostructured cantilever analyzed in this work exhibited a dimension of 40 &#181;m large and 100 &#181;m long and was fabricated with a 600 nm thick patterned quartz layer (nanopillar diameter and separation distance of 400 nm and 1 &#181;m, respectively) epitaxially grown on a 2 &#181;m thick Si device layer. The&#160;measured&#160;resonance frequency was&#160;267 kHz and the estimated quality factor, Q, of the whole mechanical structure was Q ~ 398 under low vacuum conditions. We observed the voltage-dependent linear displacement of cantilever with both techniques (i.e., AFM contact measurement and LDV). Therefore, proving that these devices can be activated through the indirect piezoelectric effect.

Epitaxial Nanostructured &#945;-Quartz Films on Silicon: From the Material to New Devices

This work presents a detailed protocol for the microfabrication of nanostructured &#945;-quartz cantilever on a Silicon-On-Insulator(SOI) technology substrate starting from the epitaxial growth of quartz film with the dip coating method and then nanostructuration of the thin film via nanoimprint lithography.

In this work, we show a detailed engineering route of the first piezoelectric nanostructured epitaxial quartz-based microcantilever. We will explain all ...

Engineering

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

Biology

Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction

Source: Matos, A. L. L., et al. <a href="https://app.jove.com/v/58224">Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator.</a> J. Vis. Exp. (2018).
This video demonstrates a dissipative microgravimetry technique to investigate the binding of proteins to lipid bilayers. Small unilamellar vesicles are added to coat the surface of the quartz sensor of a microbalance. Utilizing the calcium ion-dependent binding of the target phospholipid-binding protein to the bilayer, changes in the oscillation frequency of the sensor and the dissipation of the oscillation are monitored to determine the nature of the protein-lipid bilayer binding.

Source: Matos, A. L. L., et al. Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-lipid interactions

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

To begin plasma cleaning of the slides, purge the plasma cleaner for 5 minutes with oxygen to remove air from the lines and chamber. Arrange dry cover glass slides into a ceramic cradle. While purging, stream an inert gas over the micro cover glass slides to remove dust and particulates.
Place the cradle at the back of the plasma chamber so that the coverslips are parallel with the long edge of the chamber. Run the plasma cleaner for 15 minutes with oxygen at maximum power.
For chamber preparation, cut off the cap just below the frosted part of a 0.2-milliliter PCR tube. Paint the rim of the PCR tube with UV-activated adhesive, avoiding the inside of the tube. Gently place the PCR tube glued down in the center of a plasma-cleaned coverslip, then, place the chamber under long-wavelength UV light for 5 to 7 minutes to cure the adhesive.
To form a bilayer, add the reagents to the well. Gently shake the chambers from side to side to disrupt the SUVs, and then, incubate at 37 degrees Celsius for 20 minutes.
After incubation, rinse the bilayer 6 times with 150 microliters of SLBB, by pipetting to wash away excess lipids. Then, wash the bilayer 6 times with 150 microliters of reaction buffer, before incubating with the septins.
On the last wash step, remove the reaction buffer, leaving 75 microliters in the well. Add 25 microliters of septins diluted in SSB, and image by TIRF microscopy.

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

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