title: Circular permutation of red fluorescent proteins.
abstract: Circular permutation of fluorescent proteins provides a substrate for the design of molecular sensors. Here we describe a systematic exploration of permutation sites for mCherry and mKate using a tandem fusion template approach. Circular permutants retaining more than 60% (mCherry) and 90% (mKate) brightness of the parent molecules are reported, as well as a quantitative evaluation of the fluorescence from neighboring mutations. Truncations of circular permutants indicated essential N- and C-terminal segments and substantial flexibility in the use of these molecules. Structural evaluation of two cp-mKate variants indicated no major conformational changes from the previously reported wild-type structure, and cis conformation of the chromophores. Four cp-mKates were identified with over 80% of native fluorescence, providing important new building blocks for sensor and complementation experiments.
authors.affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.
authors.forename: Bo
authors.initials: B
authors.lastname: Shui
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Frank
authors.initials: F
authors.lastname: Lee
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Byrnes
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Chudakov
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Lukyanov
authors.forename: Holger
authors.initials: H
authors.lastname: Sondermann
authors.forename: Michael I
authors.initials: MI
authors.lastname: Kotlikoff
mesh_terms: D000595:Amino Acid Sequence; D003116:Color; D018360:Crystallography, X-Ray; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein