Directionality of light absorption and emission in representative fluorescent proteins

Protein Purification and Crystallization.

Plasmids were obtained as kind gifts from the laboratories of R. Campbell, University of Alberta, Edmonton, Canada (eGFP), D. Gadella, Univeristy of Amsterdam, Amsterdam, Holland (mTurquoise2), and R. Tsien, University of California, San Diego (mCherry), or synthesized commercially (mEos4b). FPs were expressed in Escherichia coli (strain BL21, 200-mL cultures), and harvested after induction by isopropyl β-d-1-thiogalactopyranoside (IPTG). Cells were lysed by French press, and the lysate was cleared by ultracentrifugation and loaded onto a 1-mL His-Trap column (GE Healthcare). Fast protein liquid chromatography (ÄKTA, GE Healthcare) fractions were collected during elution with a 10- to 500-mM gradient of imidazole in a 200-mM NaCl, 50-mM Tris⋅HCl (pH 7.4) buffer. Fluorescent fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fractions containing highly pure protein were pooled, and concentrated by ultrafiltration (Vivaspin 10-kDa columns, Sartorius). Crystals were grown in hanging drops, by vapor diffusion at 18 °C, as follows: mTurquoise2 (1 μL of 6 to 10 mg/mL) with 2 μL of the well solution (14% PEG 8000, 100 mM Hepes pH 8.0 to 8.5, 80 mM MgCl₂) gave rise to crystals in the P2₁2₁2₁ space group; eGFP, 5 µL of 7 mg/mL solution with 2 µL of the well solution (20% PEG 8000, 50 mM KH₂PO₄ pH 3.8), and 2 μL of 10 to 15 mg/mL with 2 μL of the well solution (17% PEG 8000, 100 mM Hepes pH 7.5, 60 mM MgCl₂); mCherry (1.5 μL of 7.5 mg/mL solution) with 2 μL of 70% well solution C2 of the JCSG++ screen (Jena Bioscience); and mEos4b (2 μL of 10 mg/mL solution) with 1.5 μL of well solution B9 of the JCSG++ screen. For X-ray measurements, crystals were mounted in a loop, flash frozen, and stored in liquid nitrogen until use. For polarization microscopy measurements, crystals were placed in a drop (5 μL) of mother liquor and sandwiched between glass coverslips sealed by an adhesive spacer (SecureSeal, Grace Biolabs). Measurements of crystals rotated along their long axis were performed by observing crystals positioned on the coverslip at an angle, because of interactions with other crystals.

X-ray Crystallography.

Diffraction datasets for eGFP, mTurquoise2, and mCherry crystals were collected in-house, on a MicroMax-007 HF Microfocus X-ray generator with a VariMax VHF ArcSec optical system (Rigaku), an AFC11 partial four-axis goniometer (Rigaku), a PILATUS 300K detector (Dectris), and a Cryostream 800 cryocooling system (Oxford Cryosystems). Diffraction datasets for mEos4b crystals were collected at 100 K on the MX 14.1 beamline operated by the Joint Berlin MX-Laboratory at the Berlin Electron Storage Ring of the Society for Synchrotron Radiation (BESSY II) in Berlin-Adlershof, Germany (44). All diffraction datasets were processed using the XDS software package (45). The crystal parameters and data collection and refinement statistics are summarized in SI Appendix, Table S1. All FP structures were solved by molecular replacement with CCP4 Molrep (46, 47), using available structures of identical proteins. The initial models were refined through several cycles of manual building using Coot and automated refinement with the software package CCP4 REFMAC5 (48). Visualizations of structural data were performed in PyMOL (49) and Chimera (50). Atomic coordinates and structure factors were deposited in the Protein Data Bank (PDB) under the codes 6YLM, 6YLN, 6YLP, 6YLQ, and 6YLS.

Optical Measurements.

Polarization microscopy measurements were performed on a modified laser scanning microscope Olympus FV1200 (SI Appendix, Fig. S1), observing at least 10 crystals of each kind. For assessment of crystal shape and orientation, image z stacks of 1-µm spacing were acquired using a 40× objective lens (UApoN 340W, 40×, numerical aperture (NA) 1.15, Olympus) and illumination wavelength at the edge of the absorption band of the observed FP, in order for the illumination to reach the full thickness of the crystal. For polarization microscopy measurements, the illuminating laser beams traveled through a Glan-laser polarizer and a 0.5-mm aperture prior to being steered into the laser scanning unit.

For xTDM determinations, the laser beams passed through a superachromatic half-wave plate (SAHWP05M-700, Thorlabs), mounted in a motorized rotating mount (PRM1/MZ8, Thorlabs), prior to entering an underfilled 10× lens (UPlanFLN 10×, NA 0.3, Olympus). Transmitted light was collected by a low NA (0.55) condenser, whose numerical aperture was reduced by an internal diaphragm to NA of ∼0.15. Before entering a photomultiplier detector, the transmitted light passed through a wavelength-specific band-pass filter (FB405-10, FL457.9-10, FL488-10, FL543.5-10, FB590-10, all Thorlabs) and a diffuser. For the red form of mEos4b, fluorescence passing through an emission filter (FELH0600, Thorlabs) was detected (instead of transmitted laser light). During measurements, polarization of the laser beam was rotated between acquisition of individual images in 10° increments over the range of 0° to 720°. Small changes in excitation light intensity with polarization modulation were noted; however, they did not affect measurements of transmittance, as they applied equally to the crystal and background segments of the analyzed images.

For mTDM determinations, restricted diameter laser beam was scanned through a 10× lens (UPlanFLN 10×, NA 0.3, Olympus). In order to ensure equal excitation of all molecular orientations present in the crystal, the excitation light was polarized linearly, parallel to the long crystal axis. Fluorescence was collected by a low NA (0.55) condenser, whose numerical aperture was reduced by an internal diaphragm to NA of ∼0.15. Before entering a photomultiplier detector, the collected fluorescence passed through an emission filter (Brightline 510/42 for mTurquoise2, 542/27 for eGFP and mEos4b, FELH0600 for mCherry and mEos4b, all Thorlabs), a polarizer mounted in a motorized rotating mount, and a diffuser. During measurements, the polarizer was rotated between acquisition of individual images in 10° increments over the range of 0° to 720°. Polarization sensitivity of the detection pathway (4.6%) was compensated for during data analysis.

FA measurements were performed in 60% glycerol/50 mM Tris solutions of FPs (10 µg/mL) in a 1-cm quartz cuvette, at 22.5 °C (as in ref. 34), using the Horiba FluoroMax4 fluorometer equipped with calcite polarizer-based fluorescence polarization module (alignment precision better than 1°). Excitation and emission slits were set to 1 and 3 nm, respectively. Signal was integrated over 1 s to 4 s. Mean values and SEs were calculated from triplicate measurements.

Microscopy Image Analysis.

Microscopy images were processed in Fiji, using publicly available tools and in-house developed macros. For xTDM determinations, the stacks of time series images acquired with different polarizations were precisely aligned by the StackReg plugin (51), and a dark detector response was subtracted. Images were then normalized by an intensity corresponding to unattenuated laser power, calculated from parts of the image not containing the crystal. The resulting transmittance images were manually segmented, and transmittance values of flat, clear parts of the observed crystals suitable for analysis were plotted as a function of polarization of the illuminating light. Values of transmittances of light polarized parallel and perpendicular to the long axis of crystal (T∥, T⊥) were obtained by fitting the transmitted light intensity data by a cosine squared function. T∥ and T⊥ were then used to derive the value of ε∥/ε⊥, (and log2(ε∥/ε⊥)), either using the simple relationship ε∥/ε⊥=log(T∥/T∥0)/log(T⊥/T⊥0) (for light of wavelengths 543 and 594 nm, of polarization purity better than 99.8%), or a more complicated formula (SI Appendix, Supplementary Text and Table S2; for wavelengths of 405, 458, and 488 nm, whose polarization purity was lower than 99.8%). Using log ratios allowed applying Gaussian statistics (generally not suitable to ratios), as well as comparing the precision of determinations of ε∥/ε⊥ spanning a large range of values. To interpret measurements of tilted crystals, the extent of rotation about the crystal long axis (crystal roll angle) was determined in Fiji by applying the angle measurement tool to vertical cross-sections of z stacks of confocal images. Z-stack cross-sections also yielded information on crystal thickness, which (when combined with protein concentration derived from the X-ray structures) allowed calculation of the molar extinction coefficients ε∥, ε⊥ (SI Appendix, Table S3). However, only the ε∥/ε⊥ ratios, derived solely from transmitted light measurements, were used for xTDM determinations.

For mTDM determinations, images of fluorescence intensity, acquired in the trans direction, with different orientations of an analyzer placed in the detection pathway, were background subtracted and manually segmented. Fluorescence intensity was then plotted as a function of the analyzer orientation. Values of fluorescence intensities polarized parallel and perpendicular to the long axis of crystal (F∥, F⊥) and their log ratio (log2(F∥/F⊥)) were obtained by fitting the fluorescence intensity data by a cosine squared function.

Mathematical Modeling.

The TDM directions with respect to the fluorophore were identified by mathematical modeling, using Mathematica (Wolfram Research). The fluorophore plane was defined by least-square fitting of coordinates of heavy atoms participating in the conjugated bond system (SI Appendix, Fig. S2). Optical measurements of the red form of mEos4b were interpreted using the published structure (PDB ID code 6GOY) aligned with the structure of the green form of mEos4b determined by us (6YLS). The TDM vector was postulated to lie within the fluorophore plane, at an angle τ with respect to a line connecting the centers of the fluorophore aromatic rings. For xTDM determinations, values of log2(ε∥/ε⊥) expected for different values of τ (SI Appendix, Fig. S3) were calculated by using a cos² relationship of the absorption rate on the angle between the polarization of the excitation light and the xTDMs present in the crystal. The log2(ε∥/ε⊥) value derived from optical measurements was compared to the log2(ε∥/ε⊥) predictions, yielding two xTDM orientations (values of τ) matching the results of crystal measurements. From the two τ values, the one consistent with measurements performed on tilted FP crystals (SI Appendix, Fig. S4) was taken to represent the TDM orientation (τ₁). An analogous procedure was used in mTDM orientation determinations, using intensities of fluorescence polarized parallel (F∥) and perpendicular (F⊥) to the long axis of crystal instead of extinction coefficients. In order to distinguish between the two τ values obtained from fluorescence polarization measurements, we used information on FA in FP solutions and directions of xTDMs.

Statistical Analysis.

All optical measurements of FP crystals were carried out on at least 10 crystals, as indicated (Table 1). Mean values and 95% CIs (±2 SEM) of values of log2(ε∥/ε⊥) and log2(F∥/F⊥) (Fig. 1 and Table 1) were used to calculate the means and 95% CIs of TDM orientations (angles τ) (Fig. 2 and Table 1). Unpaired t test was used to compare results of crystal measurements, and P value < 0.05 (marked by * in Fig. 1) was used as a statistical significance cutoff. P value < 0.001 is marked by ***. Measurements of FA were carried out in triplicates and mean values and 95% CIs (±2 SEM) were calculated (Table 1 and SI Appendix, Fig. S5).