Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure
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Contributed by Robert T. Sauer, December 21, 2004
Abstract
We designed a single-chain variant of the Arc repressor homodimer in which the β strands that contact operator DNA are connected by a hairpin turn and the α helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the sub-millisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical active-site residues in other proteins and could be a useful tool for protein design and minimization.
Footnotes
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↵ ‡ To whom correspondence should be addressed. E-mail: bobsauer{at}mit.edu.
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↵ * Present address: Fish and Richardson PC, 225 Franklin Street, Boston, MA 02110.
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↵ † Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520.
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Author contributions: R.K.T. designed research; R.K.T., B.O.C., R.A.G., and J.C.C. performed research; R.K.T., B.O.C., R.A.G., and R.T.S. analyzed data; and R.K.T., B.O.C., and R.T.S. wrote the paper.
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Abbreviation: Gdn·HCl, guanidine hydrochloride.
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Data deposition: The atomic coordinates have been deposited at the Protein Data Bank, www.pdb.org (PDB ID code 1U9P).
- Copyright © 2005, The National Academy of Sciences
