title: Computational Design of the β-Sheet Surface of a Red Fluorescent Protein Allows Control of Protein Oligomerization.
abstract: 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.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Wannier
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Moore
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Yun
authors.initials: Y
authors.lastname: Mou
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Mayo
mesh_terms: D019295:Computational Biology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D015723:Gene Library; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein