Nonlinear optical properties of photosensory core modules of monomeric and dimeric bacterial phytochromes

Abstract

Near-infrared (NIR) fluorescent proteins and optogenetic tools derived from bacterial phytochromes' photosensory core modules (PCMs) operate within the first (NIR-I) tissue transparency window under single-photon activation. Leveraging two-photon (2P) light in the second transparency window (NIR-II) for photoswitching bacterial phytochromes between Pr and Pfr absorption states offers significant advantages, including enhanced tissue penetration, spatial resolution, and signal-to-noise ratio. However, 2P photoconversion of bacterial phytochromes remains understudied. Here, we study the non-linear Pr to Pfr photoconversion's dependence on irradiation wavelength (1180-1360 nm) and energy fluence (41-339 mJ/cm²) for the PCM of DrBphP bacterial phytochrome. Our findings reveal substantially higher photoconversion efficiency for the engineered monomeric DrBphP-PCM (73%) compared to the natural dimeric DrBphP-PCM (57%). Molecular mechanical calculations, based on experimentally determined 2P absorption cross-section coefficients for the monomer (167 GM) and dimer (170 GM), further verify these results. We demonstrate both short- (SWE) and long-wavelength excitation (LWE) fluorescence of the Soret band using 405 and 810-890 nm laser sources, respectively. Under LWE, fluorescence emission (724 nm) exhibits saturation at a peak power density of 1.5 GW/cm². For SWE, we observe linear degradation of fluorescence for both DrBphP-PCMs, decreasing by 32% as the temperature rises from 19 to 38°C. Conversely, under LWE, the monomeric DrBphP-PCM's brightness increases up to 182% (at 37°C), surpassing the dimeric form's fluorescence rise by 39%. These findings establish the monomeric DrBphP-PCM as a promising template for developing NIR imaging and optogenetic probes operating under the determined optimal parameters for its 2P photoconversion and LWE fluorescence.

Keywords: DrBphP; Near‐infrared fluorescent protein (iRFP); bacterial phytochrome; long‐wavelength excitation; two‐photon photoconversion.

FIGURE 1

Light‐activatable proteins. (a) Excitation wavelength of various optogenetic proteins. (b) Structure of dimer and (c) monomer DrBphP‐PCM with highlighted PAS, GAF, and PHY domains (Protein Data Bank (PDB) ID: 4Q0J (Burgie et al., ; Burgie et al., 2016)). (d) Schematic representation of Z‐ and E‐configurations of biliverdin IXα with photoconversion conditions. (e) Single‐photon (1P) absorption spectra of Pr (brown, red hues) and Pfr (blue, green hues) states of dimeric and monomeric photosensory core modules (PCMs) (concentration of 60 μм).

FIGURE 2

Single‐photon (1P) and two‐photon (2P) conversion. (a) Absorption spectra of dimer and (b) monomer after 1P conversion to the Pfr (green line) and Pr (red line) states upon illumination with (c) 660 nm and (d) 780 nm LEDs, respectively. (e) Experimental setup for 1P Pr → Pfr, Pfr → Pr and 2P Pr → Pfr photoconversion. (f) Schematic representation of a sample cuvette illuminated by co‐directed lamp and laser beams for spectral measurements of 2P Pr → Pfr photoconversion. (g) Absorption spectra of dimer and (h) monomer under 2P Pr → Pfr photoconversion with 1180–1360 nm laser irradiation. (i) Efficiency of 2P conversion depending on laser wavelength.

FIGURE 3

Two‐photon (2P) Pr → Pfr conversion depending on energy fluence and short‐wavelength excitation (SWE) fluorescence. (a) Absorption spectra of dimer and (b) monomer depending on laser energy fluence (1250 nm). (c) Experimental and theoretical results of 2P conversion efficiency under 1250 nm irradiation, depending on laser energy fluence. (d) Experimentally measured values of fluorescence emission intensity and laser beam parameters required for the estimation of the 2P absorption cross‐section. (e) 405 nm continuous wave (CW) laser used as SWE laser source and fluorescence emission spectra of monomer depending on the laser power density. The emission spectra were recorded after passing through a 725 ± 50 nm band‐pass filter. (f) SWE fluorescence emission intensity of monomer and dimer depending on the power density (1.3–21.7 kW/cm²). (g) Schematic representation of a sample cuvette illuminated by 405 nm CW laser for spectral measurements of SWE fluorescence.

FIGURE 4

Long‐wavelength excitation (LWE) fluorescence. (a) Experimental setup for short‐wavelength excitation (SWE) and LWE fluorescence measurements. (b) Schematic representation of a sample cuvette illuminated by 810 nm pulsed laser for spectral measurements of LWE fluorescence emission. (c) LWE fluorescence emission intensity of dimer and (d) monomer under 810–890 nm light with a peak power density (PPD) of 1.57 GW/cm². (e) Pulse duration of Ti:Sapphire laser depending on laser wavelength. (f) LWE fluorescence emission intensity depending on excitation wavelength with a constant PPD of 1.57 GW/cm².

FIGURE 5

Dependence of long‐wavelength excitation (LWE) fluorescence on peak power density and temperature dependence of short‐wavelength excitation (SWE) and LWE fluorescence emission intensities. (a) LWE fluorescence emission spectra (λ _Exc = 810 nm, λ _Em = 724 nm) of dimer and (b) monomer depending on peak power density. (c) LWE fluorescence emission intensity depending on peak power density (27 MW/cm² to 1.77 GW/cm²). (d) Zoomed SWE fluorescence emission spectra (λ _Exc = 405 nm, λ _Em = 720 nm) of dimer and (e) monomer depending on the sample temperate controlled by an external heater. (f) SWE fluorescence emission intensity depending on temperature (19–38°C). (g) Zoomed LWE fluorescence emission spectra (λ _Exc = 810 nm, λ _Em = 724 nm) of dimer and (h) monomer depending on the sample temperate controlled by an external heater. (i) LWE fluorescence emission intensity depending on temperature (19–38°C).