title: Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry.
abstract: Red fluorescent indicators for calcium ion (Ca(2+)) are preferable, relative to blue-shifted alternatives, for biological imaging applications due to the lower phototoxicity, lower autofluorescent background and deeper tissue penetration associated with longer wavelength light. Accordingly, we undertook the development of a genetically encoded Ca(2+) indicator based on the popular and widely utilized Discosoma-derived red fluorescent protein, mCherry. Starting from a promising but dimly fluorescent circular permutated variant of mCherry, we first engineered a 13-fold brighter variant (cp196V1.2) through directed evolution. This bright cp196V1.2 was then used as the scaffold for creation of eight distinct libraries of potential Ca(2+) indicators via permutation at different sites within the 7th and 10th β-strands, and fusion of calmodulin and M13 to the new termini. Screening of these libraries led to the conclusion that, consistent with previous investigations of homologous fluorescent proteins, the 146-145 site in β-strand 7 is the most promising permutation site for construction of useful Ca(2+) indicators. Further rounds of directed evolution ultimately led to an indicator that exhibits a 250% change in intrinsic brightness in response to Ca(2+) and an exceptionally high affinity (Kd = 6 nM) for Ca(2+).
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors.forename: Haley J
authors.initials: HJ
authors.lastname: Carlson
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003584:Cytological Techniques; D019020:Directed Molecular Evolution; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D015220:Calcium Channels; D002147:Calmodulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein
keywords: calcium ion; calmodulin; circular permutation; fluorescent protein; imaging