This work was supported by grant NIH R01 GM097204 (APV and JDH). Funding for JHM was supplied by an HHMI Undergraduate Research Summer Fellowship.

NOTE: A diagram of the conceptual framework or workflow is described in Figure 1 for reference. The protocol can be broken down into four stages: computational or sequence based predictions, protein expression and purification, biochemical characterization, and crystallization. The examples shown analyze Shroom SD2 domains and/or Shroom-Rock complexes, but can be utilized with any protein.
1. Use Established Web-based Tools to Generate Computational Predictions of Coiled-coil Do...

<ol>
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G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Lupas, A., Van Dyke, M., Stock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+coiled+coils+from+protein+sequences.">Predicting coiled coils from protein sequences.</a> Science. 252 (5009), 1162-1164 (1991).</li><li>Offer, G., Hicks, M. R., Woolfson, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+Crick+equations+for+modeling+noncanonical+coiled+coils.">Generalized Crick equations for modeling noncanonical coiled coils.</a> J Struct Biol. 137 (1-2), 41-53 (2002).</li><li>Lupas, A. N., Gruber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+alpha-helical+coiled+coils.">The structure of alpha-helical coiled coils.</a> Adv Protein Chem. 70, 37-78 (2005).</li><li>Hernandez Alvarez, B., 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+new+expression+system+for+protein+crystallization+using+trimeric+coiled-coil+adaptors.">A new expression system for protein crystallization using trimeric coiled-coil adaptors.</a> Protein Eng Des Sel. 21 (1), 11-18 (2008).</li><li>Slabinski, L., 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=The+challenge+of+protein+structure+determination--lessons+from+structural+genomics.">The challenge of protein structure determination--lessons from structural genomics.</a> Protein Sci. 16 (11), 2472-2482 (2007).</li><li>Slabinski, L., 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=XtalPred:+a+web+server+for+prediction+of+protein+crystallizability.">XtalPred: a web server for prediction of protein crystallizability.</a> Bioinformatics. 23 (24), 3403-3405 (2007).</li><li>Haigo, S. L., Hildebrand, J. D., Harland, R. M., Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom+induces+apical+constriction+and+is+required+for+hingepoint+formation+during+neural+tube+closure.">Shroom induces apical constriction and is required for hingepoint formation during neural tube closure.</a> Curr Biol. 13 (24), 2125-2137 (2003).</li><li>Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom+regulates+epithelial+cell+shape+via+the+apical+positioning+of+an+actomyosin+network.">Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network.</a> J Cell Sci. 118 (Pt 22), 5191-5203 (2005).</li><li>Dietz, M. L., Bernaciak, T. M., Vendetti, F., Kielec, J. M., Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+actin-dependent+localization+modulates+the+evolutionarily+conserved+activity+of+Shroom+family+proteins.">Differential actin-dependent localization modulates the evolutionarily conserved activity of Shroom family proteins.</a> J Biol Chem. 281 (29), 20542-20554 (2006).</li><li>Bolinger, C., Zasadil, L., Rizaldy, R., Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+isoforms+of+drosophila+shroom+define+spatial+requirements+for+the+induction+of+apical+constriction.">Specific isoforms of drosophila shroom define spatial requirements for the induction of apical constriction.</a> Dev Dyn. 239 (7), 2078-2093 (2010).</li><li>Farber, M. J., Rizaldy, R., Hildebrand, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shroom2+regulates+contractility+to+control+endothelial+morphogenesis.">Shroom2 regulates contractility to control endothelial morphogenesis.</a> Mol Biol Cell. 22 (6), 795-805 (2011).</li><li>Das, D., 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=The+interaction+between+Shroom3+and+Rho-kinase+is+required+for+neural+tube+morphogenesis+in+mice.">The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice.</a> Biol Open. 3 (9), 850-860 (2014).</li><li>Dvorsky, R., Blumenstein, L., Vetter, I. R., Ahmadian, M. 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The protocol described here is designed to help the user identify domain boundaries within large coiled-coil proteins to facilitate their crystallization. The protocol relies on a holistic incorporation of a variety of data from computational predictions and other sources to generate a series of potential domain boundaries. These are followed by a set of biochemical experiments which are quick and inexpensive, and are used to further refine these initial hypotheses. Using this approach, the user could quickly eliminate p...

Structure determination via X-ray crystallography has made fundamental contributions to every field of modern biology; providing an atomic view of the macromolecules that support life and how they interact with one another in a variety of contexts; allowing us to understand the mechanisms that cause disease and providing opportunities to rationally design drugs to treat disease. Crystallography has long been the dominant experimental technique for determining macromolecular structure, and currently accounts for 89.3% of the structural database (www.rcsb.org). This technique has many advantages, including the potential for very high resolution, the ability to visualize macromolecules with a broad range of sizes, relatively easy data collection, and the opportunity to visualize how the macromolecule interacts with solvent as well as ligands.
Despite numerous technological improvements in recombinant protein expression1,2, purification3, and molecular biology used to generate these systems4, the single biggest obstacle in the crystallographic process remains the ability to grow diffraction quality crystals. This has been especially true for proteins which contain large coiled-coil domains. It has been estimated that as much as 5% of all amino acids are found within coiled-coils5,6, making this a very common structural feature7, yet these proteins are often more difficult to purify and crystallize than globular proteins8-10. This is further compounded by the fact that coiled-coil domains are often found within the context of a larger protein, therefore correctly predicting the boundaries of these domains is critical to avoid the inclusion of unstructured or flexible sequence that is often detrimental for crystallization.
Here we present a conceptual framework combining web-based computational analyses with experimental data from the bench, to help guide users through the initial stages of the crystallographic process including: how to select protein fragment(s) for structural studies, and how to prepare and characterize protein samples prior to crystallization attempts. We focus our analysis on two proteins containing large coiled-coil domains, Shroom (Shrm) and Rho-kinase (Rock). These proteins were chosen as they both contain coiled-coil domains and are known to form a biologically relevant complex11-16. Shroom and Rho-kinase (Rock) are predicted to contain ~200 and 680 residues of coiled-coil respectively, many portions of which have been characterized structurally17-20. The method described here provides a streamlined workflow to quickly identify fragments of coiled-coil containing protein that will be amenable for crystallization, however, the techniques described can easily be adapted for most protein or protein-complexes or modified to incorporate high-throughput approaches as available. Lastly, these methods are generally inexpensive and can be performed by users at nearly all experience levels.

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Rosetta</td>
			<td style="width:263px;">Emd Millipore</td>
			<td style="width:136px;">70954-3</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Star</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td style="width:136px;">C601003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">BL21(DE3) Codon Plus</td>
			<td style="width:263px;">Agilent Technologies</td>
			<td style="width:136px;">230245</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lysozyme</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">L3008-5GM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ni-NTA resin</td>
			<td style="width:263px;">Life Technologies</td>
			<td style="width:136px;">25216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SubtilisinA</td>
			<td style="width:263px;">Spectrum Chemical Mfg Corp</td>
			<td style="width:136px;">S1211-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well Cryschem Plate</td>
			<td>Hampton research</td>
			<td style="width:136px;">HR3-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INTELLI-PLATE &#160;96:</td>
			<td style="width:263px;">Art Robbins Instruments</td>
			<td style="width:136px;">102-0001-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 3350</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 8000</td>
			<td style="width:263px;">Hampton research</td>
			<td style="width:136px;">HR2-515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 400</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">PEG 4000</td>
			<td style="width:263px;">Hampton research</td>
			<td>HR2-605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">pcDNA3.1-Clover-mRuby2</td>
			<td style="width:263px;">Addgene</td>
			<td style="width:136px;">49089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Overnight Express Autoinduction System 1</td>
			<td style="width:263px;">Emd Millipore</td>
			<td>71300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysogeny Broth powder</td>
			<td style="width:263px;">ThermoFisher Scientific</td>
			<td>12795027&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A diagram depicting the workflow utilized in this system is shown in Figure 1 and includes three main stages. Computational analysis of the sequence is utilized to develop hypotheses about the domain boundaries of the coiled-coil protein of interest. An example of an annotated analysis of the Shrm2 SD2 domain is shown in Figure 2. In this diagram, the goal was to identify possible domain boundaries for a conserved domain at the C-terminus of the cytoskele...

Watch this Scientific Journal Video about Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins at JoVE.com

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

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 ...

The overall goal of this procedure is to rapidly identify and test possible domain boundaries within large coiled-coil proteins to improve the likelihood of finding an intrinsic fragment that will crystallize. This method can help researchers streamline the process of crystallization for large coiled-coil proteins, which can be notoriously difficult to crystallize. The advantage of this technique is that it integrates both experimental and computational methods to quickly identify and screen likely candidates for crystallization, thereby improving their chances for success.Demonstrating this technique will be Jenna Zalewski, a graduate student in my lab, and Keith O'Conor, an undergraduate researcher in my lab. To begin, combine output from established web-based tools to generate predictions of possible coiled-coil domain boundaries as described in the text protocol. After generating expression plasmids and testing their expression levels as detailed in the text, begin purification of each strain that successfully expressed Ruby tagged protein.To lyse the cells, first resuspend each thawed cell pellet in 1.5 milliliters of lysis buffer that has been supplemented with protease inhibitors. Add 30 microliters of lysozyme at a concentration of 10 milligrams per milliliter and incubate at room temperature for 20 minutes, then sonicate, following the instructions for the instrument being used. Centrifuge the sample in a tabletop centrifuge for 30 minutes at 14, 000 times G to pellet insoluble material or unlysed cells.Save both the supernatant and the pellet for subsequent analysis via SDS-PAGE. To perform nickel affinity purification, first incubate 100 microliters of the nickel NTA beads, prewashed in lysis buffer with the soluble lysate for five to 10 minutes. Invert the suspension several times to mix the beads.Centrifuge the sample at 800 times G for 30 seconds in a tabletop centrifuge to pellet the beads. Follow this by washing the pellet three to five times with lysis buffer to clean off non-specific contaminants. Finally, elute the Ruby fusion protein from the beads with lysis buffer, supplemented with one molar imidazole.Use a spectrophotometer set at 280 nanometers to measure the concentration of the Ruby fusion protein, then assess the homogeneity of the sample by loading one to five micrograms of fusion protein onto a native PAGE gel. Run the 10%native PAGE gel at four degrees Celsius for 140 minutes at 175 volts. Image the native PAGE using an imager equipped to visualize fluorescence, observing where the Ruby tagged fusion migrates in this assay.Perform limited proteolysis to identify stably folded domains by first incubating 95 microliters of Ruby-Shroom SD2 fusion with five microliters of 0.025 Subtilisin A.Sample the reaction at time points of zero, 0.5, two, five, 15, 60 and 120 minutes, removing 10 microliters from the reaction for each time point and visualizing the progress of the reaction via SDS-PAGE using a 15%acrylamide gel. Stain the gel with Coomassie blue to evaluate whether a protease-resistant species can be identified. Integrate the data from the purification efficiency, behavior in native PAGE and limited proteolysis on the partially purified mRuby2-Shroom fusion proteins into a comprehensive assessment of the overall behavior of these proteins in solution.Perform large scale expressions with two liter cultures of mRuby2-Shroom SD2 with the goal of achieving five to 20 milligrams of completely purified sample. Following cell growth, pellet the cells by centrifugation at 8, 000 times G for 10 minutes. The resulting cell pellet can be stored at minus 80 degrees Celsius until needed.Next, re-suspend the pellet in approximately eight milliliters of lysis buffer per gram of frozen cell pellet. Lyse the re-suspended cells using a homogenizer. Following cell lysis, pellet the cellular debris via centrifugation at 30, 000 times G for 30 minutes.Next, batch bind the soluble fraction using 10 milliliters of pre-washed nickel NTA resin. Pour the bead suspension into an appropriate gravity column and allow unbound proteins to drain off, then wash the resin three to five times with 40 milliliters of lysis buffer, followed by a wash with lysis buffer supplemented to one molar sodium chloride. Perform a final wash of the resin with 40 milliliters of lysis buffer supplemented with 80 millimolar imidazole.Elute the Ruby-Shroom fusion protein with 40 milliliters of lysis buffer containing one molar imidazole. Manually fractionate the eluted protein into four to 10 milliliter fractions. Confirm fractions containing Ruby-Shroom fusion protein using a 10%SDS-PAGE gel.Add one milligram of TEV protease for every 25 to 50 milligrams of fusion protein to remove the Ruby tag. Next, dialyze the appropriate fractions into TEV cleavage buffer using six to eight kilodalton molecular weight cutoff dialysis tubing. Dialyze the protein overnight at room temperature with slow stirring.Following dialysis, repeat the nickel affinity purification steps. The Shroom-SD2 domain should flow through into the unbound fraction while the Ruby tag remains bound to the resin. Using a spin concentrator, concentrate the peak fractions to approximately 10 milligrams per milliliter, then perform size exclusion chromatography analyzing peak fractions via a 12%SDS-PAGE gel.After pooling the peak fractions, dialyze the solution into crystallization buffer, then concentrate the dialyzed protein to 10 milligrams per milliliter prior to crystallization. Screen a small array of 12 conditions to identify an optimal protein concentration for crystallization trials using 24 well sitting drop crystallization trays. Perform crystallization trials using the vapor diffusion method.First, pipet 500 microliters of each of the 12 conditions into separate wells. On each pedestal, make drops that contain one microliter of well solution and one microliter of protein sample. Quickly seal the tray with clear tape, then move the tray to a suitable temperature controlled environment that is vibration-free.Examine the drops in the trays over the next three days using a microscope at up to 100X magnification. At the end of the three day period, score each drop as containing no precipitation, light precipitation, heavy precipitation or crystals. A suitable protein concentration should contain no more than six heavy precipitations.Using the determined protein concentration, screen a broad range of commercially available crystallization screens. The use of a liquid handling or crystallization robot greatly speeds up this process while also minimizing error in the drops. It requires far less protein, allowing the user to screen more conditions with a single sample.Improve the initial conditions identified from broad screens by systematically varying each of the variables within the crystallization drop using 24 well screen trays, as before. Limited proteolysis is used to identify regions of the protein that might be flexible or otherwise more accessible to protease. Shown here is a degradation pattern for an SD2 domain that is proteolytically sensitive, and another which is quite stable.Limited proteolysis was performed within the context of the Ruby fusion proteins. In this case, the linker sequence between Ruby and the SD2 domain was rapidly cleaved, as expected, but the resulting Ruby and SD2 fragments remained largely stable. Analysis by native PAGE can quickly reveal good candidates for crystallization.Four Shroom-Rock complexes with different protein batteries were run on native PAGE gels. The complex containing Rock 834 to 913 had the best behavior and crystallized readily, while the others only crystallized at degradation or failed to crystallize entirely. After watching this video, you should have a good understanding of how to develop a set of potential domain boundaries for your large coiled-coil protein and how to use the Ruby system to quickly and rapidly identify the best candidates for crystallization.While crystallography remains a time-consuming endeavor, using this technique will focus your search for proteins that will crystallize using straightforwards assays to remove poor candidates, allowing you to spend more time on the candidates with better prospects for success. While attempting this procedure, it's important to remember to include as much information as possible when selecting your possible domain boundaries. The information that you choose to select when utilizing this procedure will vary for each protein.If possible, other methods, like cell-based or biochemical assays, should be performed to ensure that the protein fragments in question still contain the function of biological interest. Although this method is focused on coiled-coil proteins, the overall framework could easily be adapted for almost any protein. Don't forget that working with certain instrumentation and biological reagents can be hazardous, and precautions, such as using gloves or wearing protective eyewear, should always be followed when performing this procedure.

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Research

JoVE Journal

Biochemistry

7.9K vistas.  University of Pittsburgh. The overall goal of this procedure is to rapidly identify and test possible domain boundaries within large coiled-coil proteins to improve the likelihood of finding an intrinsic fragment that will crystallize. This method can help researchers streamline the process of crystallization for large coiled-coil proteins, which can be notoriously difficult to crystallize. The advantage of this technique is that it integrates both experimental and computational methods to quickly identify and screen ...