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Zalewski, J.K.* ; Heber, S. ; Mo, J.H.* ; O'Conor, K.* ; Hildebrand, J.D.* ; VanDemark, A.P.*

Combining wet and dry lab techniques to guide the crystallization of large coiled-coil containing proteins.

J. Vis. Exp.:e54886 (2017)

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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.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Biochemistry ; Issue 119 ; X-ray Crystallography ; Structure Determination ; Coiled-coil ; Protein ; Rho-kinase ; Shroom; Apical Constriction; Polyacrylamide Gels; Structural Genomics; Escherichia-coli; Expression; Chromatography; Dimerization; Prediction; Binding

ISSN (print) / ISBN 1940-087X

e-ISSN 1940-087X

Journal Journal of Visualized Experiments

Quellenangaben Issue: 119, Article Number: e54886

Publisher JoVE

Publishing Place Cambridge

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Structural Biology (STB)