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. 2016 Dec 6;7:13709. doi: 10.1038/ncomms13709

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) Schematic illustrates FRET binding pairs ECFP tagged CaM and EYFP tagged myosin Va peptide containing a single IQ domain (IQ₆). (b) Left, 3³-FRET efficiency (E_A) is plotted against estimated free donor concentration (D_free). Each black symbol corresponds to data from a single cell. Right, E-FRET efficiency (E_D) is plotted as a function of estimated free acceptor concentration (A_free). The maximal 3³-FRET efficiency (E_A,max) is approximately equal to maximal E-FRET efficiency (E_D,max). (c) Cartoon illustrates FRET pairs ECFP-CaM and EYFP-tagged full length myosin Va neck domain peptide containing six IQ domains (IQ_1-6). (d) Left, 3³-FRET efficiency as a function of free donor concentration (D_free). Right, E-FRET efficiency is plotted against free acceptor concentration (A_free). In both cases, each symbol corresponds to FRET measurement from a single cell. Notice that E_A,max is ∼6-fold larger than E_D,max suggesting 6:1 donor:acceptor stoichiometry. (e) Bar-graph summary depicts maximal 3³-FRET (black) and E-FRET (red) efficiencies for binding of ECFP-CaM with various YFP-tagged truncations of myosin Va neck domain containing varying number of IQ domains as shown in cartoon below (mean±s.e.m.; n, number of cells, as labelled for each bar). (f) Experimentally determined FRET-based stoichiometry ratio (ν) follow the identity relation with the number of IQ domains within each truncation shown in e. Each symbol, mean±s.e.m.