Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes.

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 µm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 µm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

Dual Photoisomerization on Distinct Potential Energy Surfaces in a UV-Absorbing Rhodopsin.

UV-absorbing rhodopsins are essential for UV vision and sensing in all kingdoms of life. Unlike the well-known visible-absorbing rhodopsins, which bind a protonated retinal Schiff base for light absorption, UV-absorbing rhodopsins bind an unprotonated retinal Schiff base. Thus far, the photoreaction dynamics and mechanisms of UV-absorbing rhodopsins have remained essentially unknown. Here, we report the complete excited- and ground-state dynamics of the UV form of histidine kinase rhodopsin 1 (HKR1) from eukaryotic algae, using femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy, covering time scales from femtoseconds to milliseconds. We found that energy-level ordering is inverted with respect to visible-absorbing rhodopsins, with an optically forbidden low-lying S1 excited state that has Ag- symmetry and a higher-lying UV-absorbing S2 state of Bu+ symmetry. UV-photoexcitation to the S2 state elicits a unique dual-isomerization reaction: first, C13═C14 cis-trans isomerization occurs during S2-S1 evolution in <100 fs. This very fast reaction features the remarkable property that the newly formed isomer appears in the excited state rather than in the ground state. Second, C15═N16 anti-syn isomerization occurs on the S1-S0 evolution to the ground state in 4.8 ps. We detected two ground-state unprotonated retinal photoproducts, 13-trans/15-anti (all-trans) and 13-cis/15-syn, after relaxation to the ground state. These isomers become protonated in 58 µs and 3.2 ms, respectively, resulting in formation of the blue-absorbing form of HKR1. Our results constitute a benchmark of UV-induced photochemistry of animal and microbial rhodopsins.

Unraveling the Excited-State Dynamics and Light-Harvesting Functions of Xanthophylls in Light-Harvesting Complex II Using Femtosecond Stimulated Raman Spectroscopy.

Photosynthesis in plants starts with the capture of photons by light-harvesting complexes (LHCs). Structural biology and spectroscopy approaches have led to a map of the architecture and energy transfer pathways between LHC pigments. Still, controversies remain regarding the role of specific carotenoids in light-harvesting and photoprotection, obligating the need for high-resolution techniques capable of identifying excited-state signatures and molecular identities of the various pigments in photosynthetic systems. Here we demonstrate the successful application of femtosecond stimulated Raman spectroscopy (FSRS) to a multichromophoric biological complex, trimers of LHCII. We demonstrate the application of global and target analysis (GTA) to FSRS data and utilize it to quantify excitation migration in LHCII trimers. This powerful combination of techniques allows us to obtain valuable insights into structural, electronic, and dynamic information from the carotenoids of LHCII trimers. We report spectral and dynamical information on ground- and excited-state vibrational modes of the different pigments, resolving the vibrational relaxation of the carotenoids and the pathways of energy transfer to chlorophylls. The lifetimes and spectral characteristics obtained for the S1 state confirm that lutein 2 has a distorted conformation in LHCII and that the lutein 2 S1 state does not transfer to chlorophylls, while lutein 1 is the only carotenoid whose S1 state plays a significant energy-harvesting role. No appreciable energy transfer takes place from lutein 1 to lutein 2, contradicting recent proposals regarding the functions of the various carotenoids (Son et al. Chem. 2019, 5 (3), 575-584). Also, our results demonstrate that FSRS can be used in combination with GTA to simultaneously study the electronic and vibrational landscapes in LHCs and pave the way for in-depth studies of photoprotective conformations in photosynthetic systems.

The terminal phycobilisome emitter, LCM: A light-harvesting pigment with a phytochrome chromophore.

Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, L(CM). The chromophore domain of L(CM) forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in L(CM) by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of L(CM). Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.

The femtosecond-to-second photochemistry of red-shifted fast-closing anion channelrhodopsin PsACR1.

Anion channelrhodopsins (ACRs) are of great interest due to their ability to inhibit electrical signaling in optogenetic experiments. The photochemistry of ACRs is currently poorly understood and an improved understanding would be beneficial for rational design of ACRs with modified properties. Activation/deactivation of ACRs involves a series of photoreactions ranging from femtoseconds to seconds, thus real-time observation is essential to comprehend the full complexity of the photochemical processes. Here we investigate the photocycle of an ACR from Proteomonas sulcata (PsACR1), which is valuable for optogenetic applications due to the red-shifted absorption and action spectra compared to the prototype ACRs from Guillardia theta: GtACR1 and GtACR2, and the fast channel closing properties. From femto-to-submillisecond transient absorption spectroscopy, flash photolysis, and point mutations of acidic residues near the retinal Schiff base (RSB), E64, and D230, we found that the photoisomerization occurs in ∼500 fs independent of the protonation state of E64. Notably, E64 is involved in the rearrangement of the hydrogen-bond network near the RSB after photoisomerization. Furthermore, we suggest that E64 works as a primary proton acceptor during deprotonation of the RSB as has been proposed for GtACR1. Our findings allow for a deeper understanding of the photochemistry on the activation/deactivation of ACRs.

The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.

Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na(+) pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na(+) (in NaCl solution) or H(+) (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L1 (∼20 ps) → K/L2 (∼30 ns) → L/M (∼20 µs). KR2 binds a Na(+) ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na(+) unbound, Na(+) transport) and for WT KR2 in CsCl (with Na(+) unbound, H(+) transport), the extracellular Na(+) binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na(+) or H(+), does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2.

Reaction Dynamics in the Chrimson Channelrhodopsin: Observation of Product-State Evolution and Slow Diffusive Protein Motions.

Chrimson is a red-light absorbing channelrhodopsin useful for deep-tissue optogenetics applications. Here, we present the Chrimson reaction dynamics from femtoseconds to seconds, analyzed with target analysis methods to disentangle spectrally and temporally overlapping excited- and product-state dynamics. We found multiple phases ranging from ≈100 fs to ≈20 ps in the excited-state decay, where spectral features overlapping with stimulated emission components were assigned to early dynamics of K-like species on a 10 ps time scale. Selective excitation at the maximum or the blue edge of the absorption spectrum resulted in spectrally distinct but kinetically similar excited-state and product-state species, which gradually became indistinguishable on the µs to 100 µs time scales. Hence, by removing specific protein conformations within an inhomogeneously broadened ensemble, we resolved slow protein backbone and amino acid side-chain motions in the dark that underlie inhomogeneous broadening, demonstrating that the latter represents a dynamic interconversion between protein substates.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

Multicolor three-photon fluorescence imaging with single-wavelength excitation deep in mouse brain.

Multiphoton fluorescence microscopy is a powerful technique for deep-tissue observation of living cells. In particular, three-photon microscopy is highly beneficial for deep-tissue imaging because of the long excitation wavelength and the high nonlinear confinement in living tissues. Because of the large spectral separation of fluorophores of different color, multicolor three-photon imaging typically requires multiple excitation wavelengths. Here, we report a new three-photon excitation scheme: excitation to a higher-energy electronic excited state instead of the conventional excitation to the lowest-energy excited state, enabling multicolor three-photon fluorescence imaging with deep-tissue penetration in the living mouse brain using single-wavelength excitation. We further demonstrate that our excitation method results in ≥10-fold signal enhancement for some of the common red fluorescent molecules. The multicolor imaging capability and the possibility of enhanced three-photon excitation cross section will open new opportunities for life science applications of three-photon microscopy.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Membrane matters: The impact of a nanodisc-bilayer or a detergent microenvironment on the properties of two eubacterial rhodopsins.

Multi-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested the efficacy of several commonly used detergents and membrane-mimicking nanodiscs with respect to solubilization, spectral properties, thermal stability and oligomeric profile of two membrane proteins from the eubacterial rhodopsin family, green proteorhodopsin (PR) and Gloeobacter violaceus rhodopsin (GR). Good solubilization was observed for the detergents TritonX-100 and dodecylphosphocholine (DPC), but DPC in particular strongly affected the thermal stability of PR and especially GR. The least deleterious effects were obtained with n-dodecyl-ß-D-maltopyranoside (DDM) and octyl glucose neopentyl glycol (OGNG), which adequately stabilized the native oligomeric and monomeric state of PR and GR, respectively. The transition from the oligomeric to the monomeric state is accompanied by a small red-shift. Both GR and PR were rather unstable in SMA-nanodiscs, but the highest thermal stability was realized by the MSP-nanodisc environment. The size of the MSP-nanodisc was too small to fit the PR hexamer, but large enough to contain the PR monomer and GR trimer. This permitted the comparison of the photocycle of trimeric GR in a membrane-mimicking (MSP-nanodisc) and a detergent (DDM) environment. The ultrarapid early phase of the photocycle (femto- to picosecond lifetimes) showed very similar kinetics in either environment, but the slower part, initiated with proton transfer and generation of the M intermediate, proceeded faster in the nanodisc environment. The implications of our results for the biophysical characterization of PR and GR are discussed.

Photoreaction Dynamics of Red-Shifting Retinal Analogues Reconstituted in Proteorhodopsin.

Microbial rhodopsins constitute a key protein family in optobiotechnological applications such as optogenetics and voltage imaging. Spectral tuning of rhodopsins into the deep-red and near-infrared spectral regions is of great demand in such applications because more bathochromic light into the near-infrared range penetrates deeper in living tissue. Recently, retinal analogues have been successfully used in ion transporting and fluorescent rhodopsins to achieve red-shifted absorption, activity, and emission properties. Understanding their photochemical mechanism is essential for further design of appropriate retinal analogues but is yet only poorly understood for most retinal analogue pigments. Here, we report the photoreaction dynamics of red-shifted analogue pigments of the proton pump proteorhodopsin (PR) containing A2 (all- trans-3,4-dehydroretinal), MOA2 (all- trans-3-methoxy-3,4-dehydroretinal), or DMAR (all- trans-3-dimethylamino-16-nor-1,2,3,4-didehydroretinal), utilizing femto- to submillisecond transient absorption spectroscopy. We found that the A2 analogue photoisomerizes in 1.4, 3.0, and/or 13 ps upon 510 nm light illumination, which is comparable to the native retinal (A1) in PR. On the other hand, the deprotonation of the A2 pigment Schiff base was observed with a dominant time constant of 67 µs, which is significantly slower than the A1 pigment. In the MOA2 pigment, no isomerization or photoproduct formation was detected upon 520 nm excitation, implying that all the excited molecules returned to the initial ground state in 2.0 and 4.2 ps. The DMAR pigment showed very slow excited state dynamics similar to the previously studied MMAR pigment, but only very little photoproduct was formed. The low efficiency of the photoproduct formation likely is the reason why DMAR analogue pigments of PR showed very weak proton pumping activity.

Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy.

Photoprotection is fundamental in photosynthesis to avoid oxidative photodamage upon excess light exposure. Excited chlorophylls (Chl) are quenched by carotenoids, but the precise molecular origin remains controversial. The cyanobacterial HliC protein belongs to the Hlip family ancestral to plant light-harvesting complexes, and binds Chl a and ß-carotene in 2:1 ratio. We analyzed HliC by watermarked femtosecond stimulated Raman spectroscopy to follow the time evolution of its vibrational modes. We observed a 2 ps rise of the C═C stretch band of the 2Ag- (S1) state of ß-carotene upon Chl a excitation, demonstrating energy transfer quenching and fast excess-energy dissipation. We detected two distinct ß-carotene conformers by the C═C stretch frequency of the 2Ag- (S1) state, but only the ß-carotene whose 2Ag- energy level is significantly lowered and has a lower C═C stretch frequency is involved in quenching. It implies that the low carotenoid S1 energy that results from specific pigment-protein or pigment-pigment interactions is the key property for creating a dissipative energy channel. We conclude that watermarked femtosecond stimulated Raman spectroscopy constitutes a promising experimental method to assess energy transfer and quenching mechanisms in oxygenic photosynthesis.

Strong pH-Dependent Near-Infrared Fluorescence in a Microbial Rhodopsin Reconstituted with a Red-Shifting Retinal Analogue.

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. ; Biophys J. 2006 , 91 , 255 - 262 ) and 3-8 times higher than the QuasAr and PROPS voltage sensors ( Kralj , J. ; Science 2011 , 333 , 345 - 348 ; Hochbaum , D. R. ; 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. Femtosecond transient absorption spectroscopy showed that upon near-IR excitation, PR:MMAR features an unusually long fluorescence lifetime of 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.

Reaction dynamics of the chimeric channelrhodopsin C1C2.

Channelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S1) state through two conical intersections CI1 and CI2, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via CI1 and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via CI2, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection CI2 rationalizes the relatively low quantum yield of photoisomerization (30 ± 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2.

Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.