Improvement of the crystallizability and expression of an RNA crystallization chaperone

    Authors

    P. P. Ravindran; A. Heroux;J. D. Ye

    Comments

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    Abbreviated Journal Title

    J. Biochem.

    Keywords

    chaperone assisted RNA crystallography; Fab; protein engineering; shake; flask expression; surface entropy reduction; RATIONAL PROTEIN CRYSTALLIZATION; X-RAY STRUCTURES; CRYSTAL-STRUCTURE; ANGSTROM RESOLUTION; ESCHERICHIA-COLI; SYNTHETIC ANTIBODIES; CATALYTIC; DOMAIN; SECRETION SYSTEM; STRUCTURAL BASIS; RIBOZYME DOMAIN; Biochemistry & Molecular Biology

    Abstract

    Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNA-Fab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of delta C209 P4-P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of Fab-RNA complex.

    Journal Title

    Journal of Biochemistry

    Volume

    150

    Issue/Number

    5

    Publication Date

    1-1-2011

    Document Type

    Article

    Language

    English

    First Page

    535

    Last Page

    543

    WOS Identifier

    WOS:000296296500008

    ISSN

    0021-924X

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