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PEDS Advance Access originally published online on October 25, 2007
Protein Engineering Design and Selection 2007 20(11):525-534; doi:10.1093/protein/gzm046
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants

Daniel E. Strongin1, Brooke Bevis1,3, Nhi Khuong1, Maureen E. Downing2, Rita L. Strack2, Karthik Sundaram2, Benjamin S. Glick1 and Robert J. Keenan2,4

1Department of Molecular Genetics and Cell Biology 2Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA

4 To whom correspondence should be addressed. E-mail: bkeenan{at}uchicago.edu

The red fluorescent protein DsRed has been extensively engineered for use as an in vivo research tool. In fast maturing DsRed variants, the chromophore maturation half-time is ~40 min, compared to ~12 h for wild-type DsRed. Further, DsRed has been converted from a tetramer into a monomer, a task that entailed mutating approximately 20% of the amino acids. These engineered variants of DsRed have proven extremely valuable for biomedical research, but the structural basis for the improved characteristics has not been thoroughly investigated. Here we present a 1.7 Å crystal structure of the fast maturing tetrameric variant DsRed.T4. We also present a biochemical characterization and 1.6 Å crystal structure of the monomeric variant DsRed.M1, also known as DsRed-Monomer. Analysis of the crystal structures suggests that rearrangements of Ser69 and Glu215 contribute to fast maturation, and that positioning of the Lys70 side chain modulates fluorescence quantum yield. Despite the 45 mutations in DsRed.M1 relative to wild-type DsRed, there is a root-mean-square deviation of only 0.3 Å between the two structures. We propose that novel intramolecular interactions in DsRed.M1 partially compensate for the loss of intermolecular interactions found in the tetramer.

Keywords: DsRed/fast maturation/fluorescent protein/monomeric/structure

Received February 15, 2007; revised July 4, 2007; accepted August 1, 2007.

3 Present address: Whitehead Institute, Cambridge, MA 02142, USA


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