Research ArticleBIOPHYSICS

Dynamic tuning of FRET in a green fluorescent protein biosensor

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Science Advances  07 Aug 2019:
Vol. 5, no. 8, eaaw4988
DOI: 10.1126/sciadv.aaw4988
  • Fig. 1 Cartoon representation of the x-ray structure of Twitch-2B.

    (A) The fluorophores of mCerulean3 and cpVenuscd are depicted as stick structures in red. The calcium ions of the minimal TnC domain are shown as magenta spheres. The disordered linker connecting the original N and C termini of Venus is shown as a dashed line. (B) The distance between the centers of mass of the two fluorophores of Twitch-2B is depicted as a dashed line. (C) The transition dipole moments of the two fluorophores of Twitch-2B as obtained from (15) are depicted as arrows.

  • Fig. 2 Structural details of Twitch-2B.

    (A) Polar interactions between residues of the minimal TnC domain, mCerulean3, and cpVenuscd are depicted as dashed lines. The residues are shown as sticks. (B) Polar interactions mediated by residues (shown as sticks) from the linkers between mCerulean3 and the calcium-binding domain (in salmon), as well as interactions between cpVenuscd and the calcium-binding domain (in magenta). (C) Hydrophobic interactions between residues (shown as sticks) from the linkers between mCerulean3 and the calcium-binding domain (in salmon), as well as the linker between cpVenuscd and the calcium-binding domain (in magenta) with residues (in gray) from the core of the minimal TnC domain. (D and E) Close-up views of the region around N532 of Twitch-2B and of the N532F mutant of Twitch-2B (Twitch-6).

  • Fig. 3 Histograms of the paramagnetic RDC data.

    Predictions of RDC of methyl groups in the two fluorescent protein domains and TnC using the x-ray structure (in purple). The alignment tensor induced by the two dysprosium ions bound to TnC results from the translation of the tensor derived from calmodulin (see Materials and Methods). Experimental RDCs from paramagnetic NMR of Twitch-2B (in green) and Twitch-6 (in magenta). The range is reduced by a factor of 10 and 5 for Twitch-2B and Twitch-6, respectively.

  • Fig. 4 Ensembles of Twitch proteins consistent with the measured RDCs and FRET efficiencies.

    Ensembles for Twitch-2B (left) and Twitch-6 (right) contain six structures each and the largest deviating structures are shown in green and red. States that are not these extreme conformations are transparent.

  • Table 1 Ensemble selection results.

    ExperimentalEnsemble*
    RDCFRETRDCFRETQ factor
    Twitch-2B4.290.785.600.790.31
    Twitch-611.430.909.290.880.07

    *Results from the best-fitting ensembles of six structures selected with the protocol in Materials and Methods.

    †RDCs shown are the distribution ranges of the experimental and ensemble parameters. The experimental (ensemble) RDC ranges from −2.01 to 2.27 Hz (−2.50 to +3.10 Hz) for Twitch-2B and from −3.48 to +7.95 Hz (−2.64 to +6.65 Hz) for Twitch-6.

    ‡An aggregated RMS score of RDC and FRET data (see Materials and Methods).

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaaw4988/DC1

      Table S1. Data collection and refinement statistics for Twitch-2B.

      Table S2. Data collection and refinement statistics for Twitch-6.

      Fig. S1. Crystal packing of Twitch-2B.

      Fig. S2. The SAXS data of Twitch-2B show monomeric state in solution.

      Fig. S3. Donor dequenching of the calcium-bound Twitch proteins.

      Fig. S4. Emission spectrum (excitation, 432 nm) of Twitch-6 (Twitch-2B N532F) in calcium-free state (black trace) and at calcium saturation (red trace).

      Fig. S5. Experimental fluorescence absorbance and emission spectra of the isolated cpVenus and mCerulean3, respectively.

      Fig. S6. Pulse-sequence scheme for the J-modulated HMQC-TROSY experiment used to determine 1JHC couplings (Ca-loaded samples) and the corresponding 1DHC (Dy-loaded samples).

      Fig. S7. Characterization of dynamics in Twitch-2B and Twitch-6 by paramagnetic NMR.

      Fig. S8. Example of intensity peak modulation of the J-modulated HMQC-TROSY experiment from Twitch-2B acquired at 1.1 GHz.

      Data file S1. Structure_Based_FRET_Twitch-2B.xlsx (Excel file).

      Data file S2. Structure_Based_FRET_Twitch-2B.py (Python script).

    • Supplementary Materials

      The PDF file includes:

      • Table S1. Data collection and refinement statistics for Twitch-2B.
      • Table S2. Data collection and refinement statistics for Twitch-6.
      • Fig. S1. Crystal packing of Twitch-2B.
      • Fig. S2. The SAXS data of Twitch-2B show monomeric state in solution.
      • Fig. S3. Donor dequenching of the calcium-bound Twitch proteins.
      • Fig. S4. Emission spectrum (excitation, 432 nm) of Twitch-6 (Twitch-2B N532F) in calcium-free state (black trace) and at calcium saturation (red trace).
      • Fig. S5. Experimental fluorescence absorbance and emission spectra of the isolated cpVenus and mCerulean3, respectively.
      • Fig. S6. Pulse-sequence scheme for the J-modulated HMQC-TROSY experiment used to determine 1JHC couplings (Ca-loaded samples) and the corresponding 1DHC (Dy-loaded samples).
      • Fig. S7. Characterization of dynamics in Twitch-2B and Twitch-6 by paramagnetic NMR.
      • Fig. S8. Example of intensity peak modulation of the J-modulated HMQC-TROSY experiment from Twitch-2B acquired at 1.1 GHz.

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      Other Supplementary Material for this manuscript includes the following:

      • Data file S1. Structure_Based_FRET_Twitch-2B.xlsx (Excel file).
      • Data file S2. Structure_Based_FRET_Twitch-2B.py (Python script).

      Files in this Data Supplement:

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