Web Release Date: November 9,
Redesign of Protein Domains Using One-Bead-One-Compound Combinatorial Chemistry
and
Contribution from the Institute for Biomedical Research, Barcelona Science Park, 08028 Barcelona, Spain, and Department of Organic Chemistry, University of Barcelona, 08028 Barcelona Spain
Received June 1, 2007
Abstract:
A novel combinatorial strategy for the redesign of proteins based on the strength and specificity
of intra- and interprotein interactions is described. The strategy has been used to redesign the hydrophobic
core of the B domain of protein A. Using one-bead-one-compound combinatorial chemistry, 300 analogues
of the C-terminal 
-helix of the B domain, H3x, have been synthesized using a biocompatible resin and the
HMFS linker, allowing the screening to occur while the peptides were bound to the resin. The screening
was based on the ability of the H3x analogues to interact with the N-terminal helices of the B domain,
H1-H2, and retain the native B domain activity, that is binding to IgG. Eight different analogues containing
some nonconservative mutations were obtained from the library, the two most frequent of which, H3P1 and
H3P2, were studied in detail. CD analysis revealed that the active analogues interact with H1-H2. To validate
the redesign strategy the covalent modified domains H1-H2-H3P1 and H1-H2-H3P2 were synthesized and
characterized. CD and NMR analysis revealed that they had a unique, stable, and well-defined three-dimensional structure similar to that for the wild-type B domain. This combinatorial strategy allows us to
select for redesigned proteins with the desired activity or the desired physicochemical properties provided
the right screening test is used. Furthermore, it is rich in potential for the chemical modification of proteins
overcoming the drawbacks associated with the total synthesis of large protein domains.
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