title: Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging.
abstract: Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on- and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Moffitt
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: Graham T
authors.initials: GT
authors.lastname: Dempsey
authors.affiliation: Departments of Chemistry and Chemical Biology and.
authors.forename: X Sunney
authors.initials: XS
authors.lastname: Xie
authors.affiliation: Departments of Chemistry and Chemical Biology andPhysics, Harvard University,Howard Hughes Medical Institute, Cambridge, MA 02138 zhuang@chemistry.harvard.edu.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhuang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D017785:Photons; D055503:Protein Multimerization; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*; D060589:Zyxin / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C031908:H-NS protein, bacteria; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C057331:Tar protein, E coli; D014746:Vimentin; D060589:Zyxin; C040330:histone-like protein HU, bacteria
keywords: PALM; STORM; fPALM; photoconvertible; photoswitchable