Journal
JOURNAL OF PHYSICAL CHEMISTRY LETTERS
Volume 9, Issue 22, Pages 6469-6474Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jpclett.8b02780
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Funding
- Chemical Sciences Council of The Netherlands Organization for Scientific Research (NWO-CW) through a VICI grant
- Middelgroot investment grant
- Leiden University
- research programme of BioSolar Cells (BSC Core Project) [C2.9]
- Dutch Ministry of Economic Affairs
- Czech Scientific Foundation [17-01137S]
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Near-infrared (NIR)-driven rhodopsins are of great interest in optogenetics and other optobiotechnological developments such as artificial photosynthesis and deep-tissue voltage imaging. Here we report that the proton pump proteorhodopsin (PR) containing a NIR-active retinal analogue (PR:MMAR) exhibits intense NIR fluorescence at a quantum yield of 3.3%. This is 130 times higher than native PR (Lenz, M. O.; et al. Biophys J. 2006, 91, 255-262) and 3-8 times higher than the QuasAr and PROPS voltage sensors (Kralj, J.; et al. Science 2011, 333, 345-348; Hochbaum, D. R; et al. Nat. Methods 2014, 11, 825-833). The NIR fluorescence strongly depends on the pH in the range of 6-8.5, suggesting potential application of MMAR-binding proteins as ultrasensitive NIR-driven pH and/or voltage sensors. absorption spectroscopy showed that upon near-IR excitation, PR:MMAR features an unusually long 310 ps and the absence of isomerized photoproducts, consistent with the high fluorescence quantum yield. Stimulated Raman analysis indicates that the NIR-absorbing species develops upon protonation of a conserved aspartate, which promotes charge delocalization and bond length leveling due to an additional methylamino group in MMAR, in essence providing a secondary protonated Schiff base. This results in much smaller bond length alteration along the conjugated backbone, thereby conferring significant single-bond character to the C13=C14 bond and structural deformation of the chromophore, which interferes with photoinduced isomerization and extends the lifetime for fluorescence. Hence, our studies allow for a molecular understanding of the relation between absorption/emission wavelength, isomerization, and fluorescence in PR:MMAR. As acidification enhances the resonance state, this explains the strong pH dependence of the NIR emission.
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