Name: combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins
Uploaded: 2017-01-06T10:30:00
Description: 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

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>
	<li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+expression+clones+and+auto-induction+for+protein+production+in+E.+coli.">Stable expression clones and auto-induction for protein production in E. coli.</a> Methods Mol Biol. 1091, 17-32 (2014).</li><li>Studier, F. W., Moffatt, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacteriophage+T7+RNA+polymerase+to+direct+selective+high-level+expression+of+cloned+genes.">Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.</a> J Mol Biol. 189 (1), 113-130 (1986).</li><li>Chapman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification:+pure+but+not+simple.">Protein purification: pure but not simple.</a> Nature. 434 (7034), 795-798 (2005).</li><li>Gibson, D. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+interaction+of+ROCKI+with+the+switch+regions+of+RhoA.">Structural insights into the interaction of ROCKI with the switch regions of RhoA.</a> J Biol Chem. 279 (8), 7098-7104 (2004).</li><li>Mohan, 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=Structure+of+a+highly+conserved+domain+of+Rock1+required+for+Shroom-mediated+regulation+of+cell+morphology.">Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology.</a> PLoS One. 8 (12), e81075 (2013).</li><li>Mohan, 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=Structure+of+Shroom+domain+2+reveals+a+three-segmented+coiled-coil+required+for+dimerization,+Rock+binding,+and+apical+constriction.">Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction.</a> Mol Biol Cell. 23 (11), 2131-2142 (2012).</li><li>Tu, 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=Crystal+structure+of+a+coiled-coil+domain+from+human+ROCK+I.">Crystal structure of a coiled-coil domain from human ROCK I.</a> PLoS One. 6 (3), e18080 (2011).</li><li>Sievers, F., 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=Fast,+scalable+generation+of+high-quality+protein+multiple+sequence+alignments+using+Clustal+Omega.">Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.</a> Mol Syst Biol. 7, 539 (2011).</li><li>Shapiro, A. L., Vinuela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Diezel, W., Kopperschlager, G., Hofmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+procedure+for+protein+staining+in+polyacrylamide+gels+with+a+new+type+of+Coomassie+Brilliant+Blue.">An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue.</a> Anal Biochem. 48 (2), 617-620 (1972).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+limitations+of+clear-native+PAGE.">Advantages and limitations of clear-native PAGE.</a> Proteomics. 5 (17), 4338-4346 (2005).</li><li>Jungbauer, A., Hahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+chromatography.">Ion-exchange chromatography.</a> Methods Enzymol. 463, 349-371 (2009).</li><li>Yu, C. M., Mun, S., Wang, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+the+effects+of+reversible+dimerization+in+size+exclusion+chromatography.">Theoretical analysis of the effects of reversible dimerization in size exclusion chromatography.</a> J Chromatogr A. 1132 (1-2), 98-108 (2006).</li><li>Giege, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+historical+perspective+on+protein+crystallization+from+1840+to+the+present+day.">A historical perspective on protein crystallization from 1840 to the present day.</a> FEBS J. 280 (24), 6456-6497 (2013).</li><li>Lobley, A., Sadowski, M. I., Jones, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGenTHREADER+and+pDomTHREADER:+new+methods+for+improved+protein+fold+recognition+and+superfamily+discrimination.">pGenTHREADER and pDomTHREADER: new methods for improved protein fold recognition and superfamily discrimination.</a> Bioinformatics. 25 (14), 1761-1767 (2009).</li><li>Marsden, R. L., McGuffin, L. J., Jones, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+protein+domain+assignment+from+amino+acid+sequence+using+predicted+secondary+structure.">Rapid protein domain assignment from amino acid sequence using predicted secondary structure.</a> Protein Sci. 11 (12), 2814-2824 (2002).</li><li>Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., Sternberg, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Phyre2+web+portal+for+protein+modeling,+prediction+and+analysis.">The Phyre2 web portal for protein modeling, prediction and analysis.</a> Nat Protoc. 10 (6), 845-858 (2015).</li><li>Elsliger, M. A., 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+JCSG+high-throughput+structural+biology+pipeline.">The JCSG high-throughput structural biology pipeline.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 66 (Pt 10), 1137-1142 (2010).</li><li>Bach, H., 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=Escherichia+coli+maltose-binding+protein+as+a+molecular+chaperone+for+recombinant+intracellular+cytoplasmic+single-chain+antibodies.">Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies.</a> J Mol Biol. 312 (1), 79-93 (2001).</li><li>Strong, 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=Toward+the+structural+genomics+of+complexes:+crystal+structure+of+a+PE/PPE+protein+complex+from+Mycobacterium+tuberculosis.">Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.</a> Proc Natl Acad Sci U S A. 103 (21), 8060-8065 (2006).</li><li>Heras, B., Martin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-crystallization+treatments+for+improving+diffraction+quality+of+protein+crystals.">Post-crystallization treatments for improving diffraction quality of protein crystals.</a> Acta Crystallogr D Biol Crystallogr. 61 (Pt 9), 1173-1180 (2005).</li></ol>

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.

combining-wet-dry-lab-techniques-to-guide-crystallization-large

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&amp;#945;

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

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 ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

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.

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. ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

Author Spotlight: A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots 

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...