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Published November 23, 2010 | Supplemental Material + Published
Journal Article Open

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries


The longer emission wavelengths of red fluorescent proteins (RFPs) make them attractive for whole-animal imaging because cells are more transparent to red light. Although several useful RFPs have been developed using directed evolution, the quest for further red-shifted and improved RFPs continues. Herein, we report a structure-based rational design approach to red-shift the fluorescence emission of RFPs. We applied a combined computational and experimental approach that uses computational protein design as an in silico prescreen to generate focused combinatorial libraries of mCherry mutants. The computational procedure helped us identify residues that could fulfill interactions hypothesized to cause red-shifts without destabilizing the protein fold. These interactions include stabilization of the excited state through H-bonding to the acylimine oxygen atom, destabilization of the ground state by hydrophobic packing around the charged phenolate, and stabilization of the excited state by a π-stacking interaction. Our methodology allowed us to identify three mCherry mutants (mRojoA, mRojoB, and mRouge) that display emission wavelengths > 630 nm, representing red-shifts of 20–26 nm. Moreover, our approach required the experimental screening of a total of ~5,000 clones, a number several orders of magnitude smaller than those previously used to achieve comparable red-shifts. Additionally, crystal structures of mRojoA and mRouge allowed us to verify fulfillment of the interactions hypothesized to cause red-shifts, supporting their contribution to the observed red-shifts.

Additional Information

© 2010 National Academy of Sciences. Contributed by Stephen L. Mayo, September 16, 2010 (sent for review June 8, 2010). Published online before print November 8, 2010. We thank Marie L. Ary for help with the manuscript, Christina L. Vizcarra and Eric S. Zollars for help with implementation of the occluded volume solvation method, Jens Kaiser for assistance in solving crystal structures and collecting diffraction data, Pavle Niklovski for setting up crystallization screens, and Sonja Hess, Robert L.J. Graham, and Michael J. Sweredoski at the Caltech Proteome Exploration Laboratory for providing assistance with the mass spectrometry analyses. This work was supported by Defense Advanced Research Projects Agency (DARPA) Protein Design Processes. R.A.C. was supported by a fellowship from the Fonds Québécois de la Recherche sur la Nature et les Technologies. We would like to acknowledge the Gordon and Betty Moore Foundation for support of the Molecular Observatory at Caltech, and the Department of Energy and National Institutes of Health for supporting the Stanford Synchrotron Radiation Lightsource. Author contributions: R.A.C. and S.L.M. designed research; R.A.C., M.M.M., and B.D.A. performed research; B.D.A. contributed new reagents/analytic tools; R.A.C. and M.M.M. analyzed data; and R.A.C. wrote the paper.

Attached Files

Published - Chica2010p12091P_Natl_Acad_Sci_Usa.pdf

Supplemental Material - pnas.1013910107_SI.pdf


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