Computational Design of the β-Sheet Surface of a Red Fluorescent Protein Allows Control of Protein Oligomerization
Computational design has been used with mixed success for the design of protein surfaces, with directed evolution heretofore providing better practical solutions than explicit design. Directed evolution, however, requires a tractable high-throughput screen because the random nature of mutation does not enrich for desired traits. Here we demonstrate the successful design of the β-sheet surface of a red fluorescent protein (RFP), enabling control over its oligomerization. To isolate the problem of surface design, we created a hybrid RFP from DsRed and mCherry with a stabilized protein core that allows for monomerization without loss of fluorescence. We designed an explicit library for which 93 of 96 (97%) of the protein variants are soluble, stably fluorescent, and monomeric. RFPs are heavily used in biology, but are natively tetrameric, and creating RFP monomers has proven extremely difficult. We show that surface design and core engineering are separate problems in RFP development and that the next generation of RFP markers will depend on improved methods for core design.
© 2015 Wannier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: March 25, 2015; Accepted: May 21, 2015; Published: June 15, 2015. This work was supported by the Defense Advanced Research Projects Agency Protein Design Processes Program, a National Security Science and Engineering Faculty Fellowship (NSSEFF N00244-09-1-0011, N00244-09-1-0082), the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative and a National Institute of Health grant (1R21EB018579-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge the help of Melissa Lin and Shweta Bhatia for constructing some of the variants used in this study, and Alex Nisthal and Jan Kostecki for helpful discussions. Author Contributions: Conceived and designed the experiments: TW MM SM. Performed the experiments: TW. Analyzed the data: TW YM MM SM. Wrote the paper: TW YM MM SM. Data Availability: All relevant data are within the paper and its Supporting Information files. Competing interests: The authors have declared that no competing interests exist.
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