This work was supported by a grant from the National Institutes of Health (R37 HL-64159).

1. Transformation, expression, and purification of scaffold protein component
<ol>
	<li>BL21 bacterial transformation with apoE4-NT containing plasmid
	<ol>
		<li>Thaw a tube of BL21 (DE3) competent cells on ice for 10 min.</li>
		<li>Once all the ice has melted, mix gently and carefully pipette 50 &#181;L of the cells into a transformation tube on ice.</li>
		<li>Add 5 &#181;L containing 50 ng of plasmid DNA (for sequence, see Supplemental Table 1) to the cell mixture. Carefully flick the tube four or five times to mix. Do not vortex.</li>
		<li>Place the mixture on ice for 30 min.</li>
		<li>Heat shock the mixture at exactly 42 &#176;C for 10 s.</li>
		<li>Place on ice for 5 min.</li>
		<li>Pipette 950 &#181;L of S.O.C. medium into the tube at room temperature.</li>
		<li>Place the tube in a 37 &#176;C shaking incubator for 60 min with oscillation set to 250 rpm.</li>
		<li>Mix the cells thoroughly by flicking and inverting, then spread 100 &#181;L of cell solution onto a 20 mL Luria Broth + Agar selection plate treated with ampicillin at a concentration of 0.1 mg/mL.</li>
		<li>Incubate overnight at 37 &#176;C, or until visible colonies have grown.</li>
	</ol>
	</li>
	<li>Using an electronic pipettor, transfer 25 mL of sterile, NZCYM medium into a sterile 250 mL conical flask at room temperature and add ampicillin to a final working concentration of 0.1 mg/mL.</li>
	<li>Using a sterile inoculation loop, transfer one individual colony from the selection plate containing the appropriately transformed bacterial strain and add directly to the flask containing 25 mL of NZCYM media + ampicillin.</li>
	<li>Place the 25 mL of NZCYM seed culture flask into a 37 &#176;C shaking incubator with orbital oscillation set to 250 rpm. 
	NOTE: It is recommended that this seed culture be grown overnight to reach a suitable bacterial concentration (~1.5-2.0 optical density units), as measured using a spectrophotometer set to wavelength = 600 nm.</li>
	<li>Transfer 250 mL of sterile NZCYM medium into four 1 L baffled flasks and add ampicillin antibiotic to a working concentration of 0.1 mg/mL.</li>
	<li>Under sterile conditions, transfer 5 mL of saturated seed culture into each of the four 250 mL culture containing flasks and place in a 37 &#730;C shaking incubator with orbital oscillation set to 250 rpm. 
	NOTE: At this point, the culture will be referred to as an expansion culture and exponential growth will be monitored using a UV1800 spectrophotometer. Growth is allowed to proceed until the culture optical density at 600 nm (OD600) reaches a value between 0.6-0.8.</li>
	<li>Once the expansion culture has reached the desired OD600 value of 0.6-0.8, add 59.6 mg of isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to each 250 ml culture containing flask for a final concentration of 1 mM to induce protein expression. At this point, the expansion culture is referred to as the expression culture. Commence expression for a period of 5 h.</li>
	<li>At the end of the 5 h expression period, remove the four flasks from the shaking incubator and pipette 125 mL into six 250 ml centrifuge bottles (750 mL total).</li>
	<li>Balance each centrifuge bottle to ensure the total mass is within &#177;0.5 mg of one another. 
	NOTE: This step is essential to ensure safe operation of the centrifuge device.</li>
	<li>Prepare the centrifuge device by first flipping the power switch to the on position. Click the button labeled &#34;Set/Actual&#34; and adjust the parameters to &#34;JA-14&#34;, &#34;9,400 x g&#34;, 20.00 min, and 4 &#176;C using the corresponding dials.</li>
	<li>Load the six balanced centrifuge bottles into an appropriately sized centrifuge rotor and place into the centrifuge ensuring&#160;any specific safety parameters are followed. 
	NOTE: Continue this step until all bacterial cell culture has been centrifuged and supernatants collected.</li>
	<li>Filter the isolated bacterial supernatant through a vacuum filtration or equivalent apparatus fitted with a 0.45 micron filter to remove any residual debris.</li>
	<li>Heparin purification of the recombinant apoE4-NT scaffold protein 
	NOTE: The column purification procedure is conducted at room temperature.
	<ol>
		<li>Equilibrate the Hi-trap Heparin column by applying 10 column volumes (50 mL) of 10 mM sodium phosphate buffer, pH 7.2, and eluting at a rate of 5 mL/min.</li>
		<li>Apply apoE4-NT enriched medium to the column at a rate of 2.5 mL/min until the entire 1 L volume of medium has been applied and discard the flowthrough.</li>
		<li>Wash the column with 10 column volumes (50 mL) of 10 mM sodium phosphate buffer, pH 7.2, and discard the flowthrough.</li>
		<li>Elute the desired apoE4-NT protein by applying three column volumes (15 mL) of elution buffer (10 mM sodium phosphate buffer + 1.5 M NaCl, pH 7.2) to the column and collect the eluate.</li>
	</ol>
	</li>
	<li>Dialysis of apoE4-NT eluate
	<ol>
		<li>Prepare a section of dialysis tubing (dimensions of dialysis tubing used were 22 cm in length, 12 mm internal diameter, and 10,000 MWCO) by thoroughly soaking in distilled deionized (ddi) water for 10 min.</li>
		<li>Prepare 1 L of phosphate buffered saline (PBS) (10 mM sodium phosphate + 150 mM NaCl, pH 7.2) in a 1 L plastic beaker or equivalent.</li>
		<li>Using a dialysis tubing clamp, clamp one end of the soaked dialysis tubing, ensuring the clamp is secured and no liquid can escape.</li>
		<li>Insert a narrow neck funnel into the open end of the dialysis tubing and pour the 15 mL of apoE4-NT eluate into the dialysis tubing. 
		NOTE: It is imperative to check for any leaks at this step.</li>
		<li>Remove the funnel and clamp the end of the dialysis tubing with another dialysis tube clamp.</li>
		<li>Place a foam dialysis float onto one of the sealed ends of the dialysis tubing and place the assembled dialysis tubing into the beaker containing 1 L of PBS buffer.</li>
		<li>Place a magnetic stir bar into the bottom of the beaker and adjust the stirring control to &#34;low&#34;, ensuring a vortex is not formed.</li>
		<li>After dialysis has concluded, decant the retentate into a 50 mL conical tube and store at -20 &#176;C. 
		NOTE: It is recommended that this dialysis be conducted at 4 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
2. Formulation of bioactive agent containing nanodisks
<ol>
	<li>Preparation of phospholipid aliquot
	<ol>
		<li>Weigh 5 mg of an appropriate phospholipid (e.g., dimyristoylphosphatidylcholine [DMPC]) and transfer to a glass test tube.</li>
		<li>Dissolve the 5 mg of phospholipid by adding 300 &#181;L of CHCl3 and 100 &#181;L of CH3OH for a total ratio of 3:1 v/v.</li>
		<li>Evaporate the organic solvent by placing the glass test tube under a gentle stream of N2 gas for 10-15 min, such that a thin film of dried phospholipid forms along the walls of the bottom portion of the tube.</li>
	</ol>
	</li>
	<li>Lyophilization of phospholipid aliquots
	<ol>
		<li>Prepare phospholipid aliquots for lyophilization by covering the glass test tube opening with parafilm.</li>
		<li>Perforate the parafilm using a 24 G needle approximately 10-15 times.</li>
		<li>Place the perforated aliquots into an appropriate lyophilization container and ensure the rubber lid is sealed correctly.</li>
		<li>Attach the lyophilization container to the vacuum manifold located at the top of the lyophilization machine and ensure all the other valves are closed tightly.</li>
		<li>Turn on the lyophilization machine by flipping the power switch and pressing the vacuum initialization button.</li>
		<li>After the system has reached total vacuum, click the refrigeration initialization button to begin the freeze drying process. 
		NOTE: It is recommended that samples be allowed to lyophilize overnight.</li>
	</ol>
	</li>
	<li>Formulation of amphotericin-B (amp-B) nanodisks
	<ol>
		<li>Pipette 0.45 mL of PBS to the lyophilized phospholipid aliquot and vortex for ~30 s to disperse the lipid. 
		NOTE: The resulting sample will appear opaque and turbid.</li>
		<li>Pipette 50 &#181;L of a 20 mg/mL stock ampB solution (20 mg of ampB dissolved in 1 mL of dimethyl sulfoxide [DMSO], stored at -20 &#176;C in a closed amber vessel) into the dispersed phospholipid sample and vortex. 
		NOTE: When selecting a solvent to use in preparing a stock solution of the hydrophobic bioactive agent, the two overarching considerations are 1) the solubility of the bioactive agent and 2) the miscibility of the solvent with the aqueous buffer used in the formulation. While DMSO is often used15,16,17,18,19, dimethylformamide20,21 or tetrahydrofuran22 have also been successfully employed23.</li>
		<li>Pipette 0.5 mL of apoE4-NT scaffold protein (concentration of ~4 mg/mL) to the glass test tube containing the dispersed phospholipid and ampB. The final volume of the sample should be approximately 1 mL.</li>
		<li>Bath sonicate the sample at 24 &#176;C until the solution clarifies (generally 10-15 min).</li>
	</ol>
	</li>
	<li>Nanodisk centrifugation
	<ol>
		<li>Transfer the clarified ampB-nanodisk solution to a sterile 1.7 mL micro-centrifuge tube.</li>
		<li>Place the tube into a tabletop micro-centrifuge rotor with the addition of a balance tube placed directly opposite.</li>
		<li>Tighten the rotor cap and close the centrifuge lid.</li>
		<li>Program the centrifuge to spin for 10 min at ~11,000 x g using the corresponding dials located on the front of the micro-centrifuge unit. 
		NOTE: At this point, a pellet may be visible. This pellet consists of unincorporated phospholipid and/or ampB.</li>
		<li>Remove the supernatant and transfer it to another clean 1.7 mL micro-centrifuge tube.</li>
	</ol>
	</li>
	<li>Dialysis of ampB-nanodisk sample
	<ol>
		<li>Prepare a section of dialysis tubing (dimensions of dialysis tubing used were 3 cm in length, 16 mm internal diameter, and 10,000 MWCO) by thoroughly soaking in ddi water for 10 min.</li>
		<li>Prepare a 1 L PBS buffer solution (10 mM sodium phosphate + 150 mM NaCl, pH 7.2) in a 1 L plastic beaker or equivalent.</li>
		<li>Using a dialysis tubing clamp, clamp one end of the soaked dialysis tubing, ensuring the clamp is secured and no liquid can escape.</li>
		<li>Insert a narrow neck funnel into the open end of the dialysis tubing and transfer the ampB-nanodisk sample into the dialysis tubing.</li>
		<li>Remove the funnel and clamp the end of the dialysis tubing with another dialysis tube clamp.</li>
		<li>Place a foam dialysis float onto one of the sealed ends of the dialysis tubing and place the assembled dialysis tubing into the beaker containing the 1 L of PBS buffer.</li>
		<li>Place a magnetic stir bar into the bottom of the beaker and adjust the stirring control to &#34;low&#34;, ensuring a vortex is not formed. Allow dialysis to continue overnight at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Spectral analysis of ampB-nanodisk samples
<ol>
	<li>Spectrophotometer initialization followed by auto blank
	<ol>
		<li>Turn on the spectrophotometer by flipping the power switch and connect to a corresponding support computer by pressing the button labeled &#34;PC Control&#34;.</li>
		<li>On the support computer, open the software labeled &#34;UVProbe 2.61&#34; and connect to the spectrophotometer by clicking the button labeled &#34;Connect&#34; in the bottom left corner.</li>
		<li>Click the button labeled Spectrum on the top toolbar within the UVProbe software.</li>
		<li>Click the button labeled Method on the top toolbar.</li>
		<li>Click on the Measurement tab and input &#34;500&#34; into the Start textbox located under &#34;Wavelength Range (nm)&#34; and &#34;300&#34; into the &#34;End&#34; textbox.</li>
		<li>Click the drop-down menu next to the tab labeled Scan Speed and set it to Medium.</li>
		<li>Prepare a sample blank by transferring 1 ml of DMSO into two quartz cuvettes (QS 1.000).</li>
		<li>Load both cuvettes into the respective sample ports of the spectrophotometer, click Autoblank, and record a spectrum from 300-500 nm by clicking Start in the bottom left corner.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of ampB standard
	<ol>
		<li>Prepare 20 &#181;g/mL ampB standard by removing the cuvette from the front sample port and adding 20 &#181;L of a 1 mg/mL ampB stock solution (20 &#181;g of ampB total).</li>
		<li>Load the cuvette back into the sample port of the spectrophotometer and click Start to record the sample absorbance.</li>
		<li>Remove the cuvette from the sample port and decant the liquid contents into an appropriately labeled waste container.</li>
		<li>Thoroughly rinse the cuvette with three washes of deionized water followed by three washes with 70% ethanol.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of disrupted ampB-nanodisk sample
	<ol>
		<li>Prepare a disrupted ampB-nanodisk sample (containing 20 &#181;g/mL as ampB) by pipetting 20 &#181;L of a 1 mg/mL ampB-nanodisk stock into 1 mL of DMSO. Incubate for at least 1 min prior to recording the spectrum.</li>
		<li>Load the cuvette into the front sample port of the spectrophotometer and click Start to record the sample absorbance.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of a PBS buffer blank
	<ol>
		<li>Prepare a PBS buffer blank by transferring 1 mL of PBS buffer into two quartz cuvettes (QS 1.000).</li>
		<li>Load the cuvettes into the respective sample ports of the spectrophotometer, click Autoblank, and record the spectrum from 300-500 nm.</li>
	</ol>
	</li>
	<li>Preparation and spectral analysis of a non-disrupted ampB-nanodisk sample
	<ol>
		<li>Prepare a non-disrupted ampB-nanodisk sample (containing 20 &#181;g/mL as ampB) by removing the cuvette from the front sample port and introducing 20 &#181;L of a 1 mg/mL ampB-nanodisk sample into the PBS buffer.</li>
		<li>Load the cuvette back into the sample port of the spectrophotometer, click Start, and record the sample spectrum. 
		&#8203;NOTE: All chemical waste should be disposed of in an appropriately labeled waste container following accepted guidelines.</li>
	</ol>
	</li>
</ol>
4. Yeast viability assay analysis
NOTE: Yeast viability assays were performed in order to evaluate the biological activity of ampB and determine whether the process of formulation or incorporation into nanodisks, affected its yeast growth inhibition activity.
<ol>
	<li>Formulate two ampB-nanodisk samples (final volume = 1 mL) to contain 1 mg of ampB per 5 mg of DMPC and 0.1 mg of ampB per 5 mg of DMPC following the previously described method (2.3-2.4.1). 
	NOTE: To make a 1 mg/mL and 0.1 mg/mL ampB per 5 mg of DMPC, pipette 50 &#181;L and 5 &#181;L, respectively, of a 20 mg/mL ampB in DMSO stock into each sample vial.</li>
	<li>Preparation of a saturated yeast culture
	<ol>
		<li>Transfer 25 mL of sterile Yeast Extract-Peptone-Dextrose (YPD) medium into a sterile 50 mL conical tube.</li>
		<li>Use a sterile inoculation loop to transfer a single Saccharomyces cerevisiae (BY4741) colony into the 25 mL of YPD medium.</li>
		<li>Culture the yeast in a shaking incubator set at 30 &#176;C, with oscillation set to 200 rpm for 18 h.</li>
	</ol>
	</li>
	<li>Preparation of individual growth inhibition assay samples
	<ol>
		<li>Transfer 5 mL of sterile YPD medium into 30 sterile 13 mL culture tubes.</li>
		<li>Treat three sets of culture tubes by adding 20, 10, and 5 &#181;L of the 1 mg/mL ampB-nanodisk sample, representing 20, 10, and 5 &#181;g of ampB, respectively.</li>
		<li>Treat three sets of culture tubes by adding 20, 10, and 5 &#181;l of the 0.1 mg/mL ampB-nanodisk sample, representing 2, 1, and 0.5 &#181;g of ampB, respectively.</li>
		<li>Treat four sets of culture tubes by adding 20 &#181;L of PBS, DMSO, control rHDL (no ampB), or 20 &#181;g of ampB in DMSO. 
		NOTE: Each set of culture tubes represents an independent replicate. Therefore, following this methods results in an overall n value of n = 3.</li>
	</ol>
	</li>
	<li>Yeast viability assay initialization
	<ol>
		<li>Initiate the experiment by inoculating each sample with 100 &#181;L (2% vol/vol) of the saturated yeast culture</li>
		<li>Culture the yeast using a shaking incubator set to 30 &#176;C, with oscillation set to 200 rpm for a maximum of 18 h.</li>
	</ol>
	</li>
	<li>Yeast viability measurements
	<ol>
		<li>Turn on the spectrophotometer by flipping the power switch, then click the button labeled Go To WL and set the value to 600 nm.</li>
		<li>Transfer 1 mL of sterile YPD medium into two plastic cuvettes, load into the respective sample ports on the spectrophotometer, and click Autoblank.</li>
		<li>Transfer 1 mL of each sample into a plastic cuvette, making sure to change cuvettes each time and load the cuvette into the front sample port of the spectrophotometer.</li>
		<li>Measure and record each sample&#39;s optical density at 600 nm and dispose of each expended cuvette into a properly labeled biohazard waste container.</li>
	</ol>
	</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+HDL+nanoparticles+delivering+docetaxel+and+CpG+for+chemoimmunotherapy+of+colon+adenocarcinoma.">Synthetic HDL nanoparticles delivering docetaxel and CpG for chemoimmunotherapy of colon adenocarcinoma.</a> International Journal of Molecular Sciences. 21 (5), 1777 (2020).</li><li>Duivenvoorden, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statin-loaded+reconstituted+high-density+lipoprotein+nanoparticle+inhibits+atherosclerotic+plaque+inflammation.">A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation.</a> Nature Communications. 5, 3065 (2014).</li><li>Hargreaves, P. L., Nguyen, T. S., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+studies+of+amphotericin+B+solubilized+in+nanoscale+bilayer+membranes.">Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes.</a> Biochimica et Biophysica Acta. 1758 (1), 38-44 (2006).</li><li>Tufteland, M., Pesavento, J. B., Bermingham, R. L., Hoeprich Jr, P. D., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+stabilized+amphotericin+B+nanodisks.">Peptide stabilized amphotericin B nanodisks.</a> Peptides. 28 (4), 741-746 (2007).</li><li>Tufteland, M., Ren, G., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodisks+derived+from+amphotericin+B+lipid+complex.">Nanodisks derived from amphotericin B lipid complex.</a> Journal of Pharmaceutical Sciences. 97 (10), 4425-4432 (2008).</li><li>Nguyen, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphotericin+B+induces+interdigitation+of+apolipoprotein+stabilized+nanodisk+bilayers.">Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers.</a> Biochimica et Biophysica Acta. 1778 (1), 303-312 (2008).</li><li>Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobiotechnology+applications+of+reconstituted+high+density+lipoprotein.">Nanobiotechnology applications of reconstituted high density lipoprotein.</a> Journal of Nanobiotechnology. 8, 28 (2010).</li><li>Lalefar, N. R., Witkowski, A., Simonsen, J. B., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt3a+nanodisks+promote+ex+vivo+expansion+of+hematopoietic+stem+and+progenitor+cells.">Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells.</a> Journal of Nanobiotechnology. 14 (1), 66 (2016).</li><li>Crosby, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-CD20+single+chain+variable+antibody+fragment-apolipoprotein+A-I+chimera+containing+nanodisks+promote+targeted+bioactive+agent+delivery+to+CD20-positive+lymphomas.">Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.</a> Biochemistry and Cell Biology. 93 (4), 343-350 (2015).</li><li>Ghosh, M., Ren, G., Simonsen, J. B., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid+nanodisks+as+an+siRNA+delivery+vehicle.">Cationic lipid nanodisks as an siRNA delivery vehicle.</a> Biochemistry and Cell Biology. 92 (3), 200-205 (2014).</li><li>Fox, C. A., Ellison, P., Ikon, N., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+transformation+of+cardiolipin+nanodisks.">Calcium-induced transformation of cardiolipin nanodisks.</a> Biochimica et Biophysica Acta. Biomembranes. 1861 (5), 1030-1036 (2019).</li><li>Fox, C. A., Lethcoe, K., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-induced+release+of+cytochrome+c+from+cardiolipin+nanodisks:+Implications+for+apoptosis.">Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis.</a> Biochimica et Biophysica Acta Biomembranes. 1861 (12), 183722 (2021).</li><li>Fox, C. A., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+binding+assay+reveals+doxorubicin+preference+for+DNA+versus+cardiolipin.">Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.</a> Analytical Biochemistry. 594, 113617 (2020).</li><li>Fox, C. A., Romenskaia, I., Dagda, R. K., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+nanodisks+confer+protection+against+doxorubicin-induced+mitochondrial+dysfunction.">Cardiolipin nanodisks confer protection against doxorubicin-induced mitochondrial dysfunction.</a> Biochimica et Biophysica Acta Biomembranes. 1864 (10), 183984 (2022).</li></ol>

The authors have nothing to disclose.

Formulation of a bioactive agent containing nanodisks provide&#160;a convenient method to solubilize otherwise insoluble hydrophobic compounds. Because the product bioactive agent nanodisks are fully soluble in aqueous media, they provide a useful delivery method for a wide range of hydrophobic molecules (Table 1). These include small molecules, natural and synthetic drugs, phytonutrients, hormones, etc. The formulation strategy usually follows a standard protocol that must take into consideration the so...

Nascent discoidal high density lipoproteins (HDLs) are naturally occurring progenitors of the far more abundant spherical HDL present in the human circulatory system. These nascent particles, also referred to as pre-&#223; HDL, possess unique and distinctive structural properties1. Indeed, rather than existing as a spheroidal particle, nascent HDLs are disk-shaped. Extensive structural characterization studies on natural and reconstituted discoidal HDLs have revealed that they are comprised of a phospholipid bilayer whose perimeter is circumscribed by an amphipathic exchangeable apolipoprotein (apo), such as apoA-I. In human lipoprotein metabolism, circulating nascent HDLs accrue lipids from peripheral cells and mature into spherical HDLs in a process that is dependent upon key protein mediators, including the ATP binding cassette transporter A1 and lecithin:cholesterol acyltransferse2. This process represents a critical component of the reverse cholesterol transport pathway that is considered to be protective against heart disease. Armed with this knowledge and the ability to reconstitute discoidal HDLs, researchers have employed these particles as a therapeutic intervention to treat atherosclerosis3. In essence, the infusion of reconstituted HDL (rHDL) into patients promotes cholesterol efflux from plaque deposits and returns it to the liver for conversion to bile acids and excretion from the body. Several biotechnology/pharmaceutical companies are pursuing this treatment strategy4.
At the same time, the ability to generate these particles in the laboratory has sparked a flurry of research activities that has led to novel applications and new technologies. One prominent application involves the use of rHDL particles as a miniature membrane to house transmembrane proteins in a native-like environment5. To date, hundreds of proteins have been successfully incorporated into discoidal rHDL, and research has demonstrated that these proteins retain both native conformation and biological activity as receptors, enzymes, transporters, etc. These particles, referred to as &#34;nanodiscs&#34;, have also been shown to be amenable to structural characterization, often at high resolution6. This approach to investigations of transmembrane proteins is recognized as superior to studies with detergent micelles or liposomes and, as a result, is rapidly advancing. It is important to recognize that two distinct methods have been reported that are capable of forming an rHDL. The &#34;cholate dialysis&#34; method13 is popular for applications related to the incorporation of transmembrane proteins in the rHDL bilayer5. Essentially, this method of formulation involves mixing a bilayer forming phospholipid, a scaffold protein, and the transmembrane protein of interest in a buffer containing the detergent sodium cholate (or sodium deoxycholate; micelle molecular weight [MW] of 4,200 Da). The detergent effectively solubilizes the different reaction components, permitting the sample to be dialyzed against buffer lacking detergent. During the dialysis step, as the detergent is removed from the sample, an rHDL spontaneously forms. When this approach is used to entrap a transmembrane protein of interest, the product particles have been termed nanodiscs5. Attempts to use this method to incorporate small molecule hydrophobic bioactive agents (MW &#60;1,000 Da), however, have been largely unsuccessful. Unlike transmembrane proteins, small molecule bioactive agents are able to escape from the dialysis bag along with the detergent, greatly decreasing their incorporation efficiency into rHDLs. This problem was solved by omitting detergents from the formulation mixture14. Instead, the components are added to an aqueous buffer sequentially, beginning with the bilayer forming lipid, forming a stable bioactive agent containing rHDL, referred to as a nanodisk. Others have used rHDL for the incorporation and transport of in vivo imaging agents7. More recently, specialized rHDL comprised of an apolipoprotein scaffold and the anionic glycerophospholipid, cardiolipin, have been employed in ligand binding studies. These particles provide a platform for studies of the interaction of cardiolipin with various water soluble ligands, including calcium, cytochrome c, and the anticancer agent doxorubicin8.
The focus of the present study is on the formulation of rHDL that possess a stably incorporated hydrophobic bioactive agent (i.e. nanodisk). The ability of these agents to integrate into the lipid milieu of discoidal rHDL particles effectively confers them with aqueous solubility. As such, nanodisks have the potential for in vivo therapeutic applications. When formulating nanodisks, specific incubation/reaction conditions are required to successfully incorporate discrete hydrophobic bioactive agents into the product particle, and the goal of this report is to provide detailed practical information that can be used as a foundational template for creating novel nanodisk particles for specific applications. Thus, in the context of this manuscript the terms nanodisc and nanodisk are not interchangeable. Whereas nanodisc refers to an rHDL formulated to contain a transmembrane protein embedded in its lipid bilayer5, the term nanodisk refers to an rHDL formulated to incorporate low molecular weight (&#60; 1,000 Da) hydrophobic bioactive agents, such as amphotericin B14.
A variety of methods are available for the acquisition of suitable scaffold proteins. It is possible to purchase scaffold proteins from manufacturers [e.g. apoA-I (SRP4693) or apoE4 (A3234)], however, the cost may be a limiting factor. A preferred approach is to express recombinant scaffold proteins in Escherichia coli. Protocols are published for human apoA-I9, apoE410, as well as the insect hemolymph protein apolipophorin-III11. For the purpose of the experiments described herein, recombinant human apoE4 N-terminal (NT) domain (amino acids 1-183) was used. The nucleotide sequence encoding human apoE4-NT was synthesized and inserted into a pET-22b (+) expression vector directly adjacent to the vector-encoded pelB leader sequence. This construct leads to the expression of a pelB leader sequence-apoE4-NT fusion protein. Following protein synthesis, the bacterial pelB leader sequence directs the newly synthesized protein to the periplasmic space where leader peptidase cleaves the pelB sequence. The resultant apoE4-NT protein, with no sequence tags or tails, subsequently escapes the bacteria and accumulates in the culture medium11,12, simplifying downstream processing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>Cayman Chemical Company</td>
			<td>11636</td>
			<td>ND Formulation &#38; Standard Preparation</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP17925</td>
			<td>Transformation &#38; Expansion</td>
		</tr>
		<tr>
			<td>ApoE4-NT Plasmid</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Baffled Flask</td>
			<td>New Brunswick Scientific</td>
			<td>N/A</td>
			<td>Expansion &#38; Expression</td>
		</tr>
		<tr>
			<td>BL21 competent E coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Centrifuge bottles</td>
			<td>Nalgene</td>
			<td>3140-0250</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>G607-4</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td>Standard Prepartation</td>
		</tr>
		<tr>
			<td>Dymyristoylphosphatidylcholine</td>
			<td>Avanti Lipids</td>
			<td>850345P</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Erlenmeyer flask</td>
			<td>Bellco Biotechnology</td>
			<td>N/A</td>
			<td>Expansion &#38; Expression</td>
		</tr>
		<tr>
			<td>Falcon Tubes</td>
			<td>Sarstedt Ag &#38; Co</td>
			<td>D51588</td>
			<td>Yeast Viability Assay</td>
		</tr>
		<tr>
			<td>Glass borosilicate tubes</td>
			<td>VWR</td>
			<td>47729-570</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>GraphPad (Software)</td>
			<td>Dotmatics</td>
			<td>N/A</td>
			<td>Yeast Viability Assay</td>
		</tr>
		<tr>
			<td>Heated Sonication Bath</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>ND Formulaton</td>
		</tr>
		<tr>
			<td>Heating and Nitrogen module</td>
			<td>Thermo Scientific</td>
			<td>TS-18822</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP (5 mL)</td>
			<td>GE Healthcare</td>
			<td>17-0407-03</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP1755</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>J-25 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>J325-IM-2</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>JA-14 Rotor</td>
			<td>Beckman Coulter</td>
			<td>339247</td>
			<td>Expression</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7755030</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>Praxair</td>
			<td>UN1066</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>NZCYM media</td>
			<td>RPI Research Products</td>
			<td>N7200-1000.0</td>
			<td>Expansion &#38; Expression</td>
		</tr>
		<tr>
			<td>Pet-22B vector</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td>Transformation</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875718</td>
			<td>Transformation &#38; Expansion</td>
		</tr>
		<tr>
			<td>Quartz Cuvettes</td>
			<td>Fisher Brand</td>
			<td>14385 928A</td>
			<td>Spectral Analysis</td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>New Brunswick Scientific</td>
			<td>M1344-0004</td>
			<td>Transformation, Expansion, &#38; Expression</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer Buoys</td>
			<td>Thermo Scientific</td>
			<td>66430</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing</td>
			<td>Thermo Scientific</td>
			<td>88243</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP329-500</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Spectra/POR Weighted Closures</td>
			<td>Spectrum Medical Industries</td>
			<td>132736</td>
			<td>Purification</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shimadzu UV-1800</td>
			<td>220-92961-01</td>
			<td>spectral analysis</td>
		</tr>
		<tr>
			<td>Tabletop Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>366816</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>UVProbe 2.61 (Software)</td>
			<td>Shimadzu</td>
			<td>N/A</td>
			<td>Spectral Analysis</td>
		</tr>
		<tr>
			<td>Vacuum filter</td>
			<td>Millipore</td>
			<td>9004-70-0</td>
			<td>Expression &#38; Purification</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>GAST Manufacturing Inc</td>
			<td>DOA-P704-AA</td>
			<td>Expression &#38; Purification</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td>ND Formulation</td>
		</tr>
		<tr>
			<td>Yeast</td>
			<td>N/A</td>
			<td>BY4741</td>
			<td>Yeast Viability Assay</td>
		</tr>
		<tr>
			<td>Yeast Extract-Peptone-Dextrose</td>
			<td>BD</td>
			<td>242820</td>
			<td>Yeast Viability Assay</td>
		</tr>
	</tbody>
</table>

formulation and characterization of bioactive agent containing nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Bioactive agent nanodisk formulation process 
In the ampB-nanodisk formulation procedure described, the reaction is considered complete when the sample appearance transitions from turbid to clear (Figure 1). This change indicates that nanodisks have formed and that the bioactive agent has been solubilized. Oftentimes, bioactive agents absorb light in the visible wavelength region (e.g., ampB, curcumin, lutein, coenzyme Q10) and, in these cases, the sample ado...

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Formulation and Characterization of Bioactive Agent Containing Nanodisks

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

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950 Views.  University of Nevada. Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner. 

Video: Formulation and Characterization of Bioactive Agent Containing Nanodisks

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...