Kyril M. Solntsev

Topochemistry and photomechanical effects in crystals of green fluorescent protein-like chromophores: effects of hydrogen bonding and crystal packing.

To obtain insight into the effects of the environment on the photophysics and photochemistry of the green fluorescence protein (GFP), eight crystal structures of six synthetic aryl-substituted analogues (2-fluoro, 2-methyl, 3-hydroxy, 3-methoxy, 2,4-dimethyl and 2,5-dimethyl) of the GFP chromophore (4-hydroxy-benzylidenedimethylimidazolinone) were determined and correlated with their two-dimensional steady-state and time-resolved solid-state excitation-emission spectra. The stacking between the molecules greatly affected the emission energy and the lifetime of the emission of the chromophore, implying that pi-pi interactions could be critical for the photophysics of GFP. The reaction pathways were dependent on the excitation energy, resulting either in [2 + 2] photodimerization at the bridging double bond (UV excitation) or flipping of the imidazolone ring (visible excitation). The meta-hydroxy chromophore (3-HOBDI) was the only GFP-chromophore analogue that was obtained as more than one stable polymorph in the pure state thus far. Due to the asymmetric substitution with hydrogen bond donors and acceptors, 3-HOBDI is tetramorphic, the forms showing distinctly different structure and behavior: (1) while one of the polymorphs (3-HOBDI-A), having multilayer structure with alternating stereochemistry of linear hydrogen-bonded motifs, undergoes photodimerization under UV light, (2) another (3-HOBDI-C), which has dimeric head-to-tail structure, shows Z-to-E isomerization via tau-one-bond flip of the imidazolone ring by excitation in the visible region. X-ray diffraction analysis of a partially reacted single crystal of 3-HOBDI-C provided the first direct evidence of tau-one-bond flip occurring in a GFP-like compound. Moreover, the cooperative action of the photodimerization of 3-HOBDI-A appears as a photomechanical effect of unprecedented magnitude for a single crystalline specimen, where photoexcited single crystals bend to more than 90 degrees without breaking.

Activation and Tuning of Green Fluorescent Protein Chromophore Emission by Alkyl Substituent-Mediated Crystal Packing

O-Alkyl synthetic analogues of the green fluorescent protein chromophore are nonfluorescent in solution but demonstrate a bright emission in the crystalline state. Three-dimensional steady-state and time-resolved emission spectroscopies in the solid state, as well as single-crystal X-ray diffraction, reveal the nature of complex emission in the crystals. A hypsochromic emission shift with an increase of the alkyl size is determined by the monomer-aggregate emission ratio.

Meta and para effects in the ultrafast excited-state dynamics of the green fluorescent protein chromophores

Femtosecond transient absorption and fluorescence upconversion experiments have been performed to investigate the photoinduced dynamics of the meta isomer of the green fluorescent protein chromophore, m-HBDI, and its O-methylated derivative, m-MeOBDI, in various solvent mixtures at neutral, acidic, and basic pH. The para isomer, p-HBDI, and its O- and N-methylated derivatives, p-MeOBDI and p-HBDIMe(+), were also studied for comparison. In all cases, fast quenching of the excited S1 state by internal conversion (IC) to the ground state was observed. In the para compounds, IC, presumably promoted by the internal twisting, arises in <1 ps. A similar process takes place in the meta compounds in nonaqueous solvents but with notably slower kinetics. In aqueous solutions, the meta compounds undergo ultrafast intermolecular excited-state proton transfer that competes with isomerization.

Excited-state reversible geminate reaction. I. Two different lifetimes

We obtain an accurate long-time solution for the diffusive kinetics of an excited geminate pair dissociating reversibly in solution, where the excited-state lifetimes of the bound and unbound states may be nonequal. We analyze the long-time asymptotic behavior, finding a transition between two types of behaviors. In region A, the excited bound pair decays as t−3/2 exp(−t/τ0′), where τ0′ is the lifetime of the unbound state. In region AB, the decay is a pure exponential. At the critical value of the parameters where the transition occurs, we find a t−1/2 exp(−t/τ0′) decay. These predictions are tested against an exact numerical solution of the corresponding coupled partial differential equations. For region A, we supply an experimental example involving time-resolved excited-state proton transfer from 5-cyano-2-naphthol to dimethyl sulfoxide.

Isomerization in Fluorescent Protein Chromophores Involves Addition/Elimination

The green fluorescent protein (GFP) chromophore undergoes both photochemical and thermal isomerizations. Typically, the Z form is more stable and undergoes photochemical conversion to the E form followed by thermal reversion over a period of seconds or minutes. Although the mechanism of the thermal reversion has been the subject of some investigations, the surprisingly low activation energy for this process has not sparked any controversy. We now show that the chromophore is surprisingly stable in both E and Z forms and that the facile thermal reversion is the result of a novel nucleophilic addition/elimination mechanism. This observation may have implications for the intervention of such processes, as well as blinking and kindling, in fluorescent proteins.

Fluorescence imaging using synthetic GFP chromophores

Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.

Chemically modulating the photophysics of the GFP chromophore.

There is growing interest in engineering the properties of fluorescent proteins through modifications to the chromophore structure utilizing mutagenesis with either natural or unnatural amino acids. This entails an understanding of the photophysical and photochemical properties of the modified chromophore. In this work, a range of GFP chromophores with different alkyl substituents are synthesized and their electronic spectra, pH dependence, and ultrafast fluorescence decay kinetics are investigated. The weakly electron donating character of the alkyl substituents leads to dramatic red shifts in the electronic spectra of the anions, which are accompanied by increased fluorescence decay times. This high sensitivity of electronic structure to substitution is also characteristic of some fluorescent proteins. The solvent viscosity dependence of the decay kinetics are investigated, and found to be consistent with a bimodal radiationless relaxation coordinate. Some substituents are shown to distort the planar structure of the chromophore, which results in a blue shift in the electronic spectra and a strong enhancement of the radiationless decay. The significance of these data for the rational design of novel fluorescent proteins is discussed.

Chromophore photoreduction in red fluorescent proteins is responsible for bleaching and phototoxicity.

Red fluorescent proteins (RFPs) are indispensable tools for deep-tissue imaging, fluorescence resonance energy transfer applications, and super-resolution microscopy. Using time-resolved optical spectroscopy this study investigated photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, a new transient absorption intermediate was observed, which decays on a microsecond–millisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68–1.72 eV (λmax = 720–740 nm). On the basis of electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction. A doubly charged state that is not stable in the isolated (gas phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.

Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein Chromophore

Cis-trans photoisomerization is proposed as a key process in the photoswitching of some photoactivatable fluorescent proteins. Here we present ultrafast fluorescence measurements of the model GFP chromophore (HBDI) in the cis state and in a mixture of the cis and trans states. Our results demonstrate that the mean lifetimes of the cis and trans states are remarkably similar. Therefore, the specific isomer of the chromophore cannot be solely responsible for the different photophysics of the bright and dark states of photoactive proteins, which must therefore be due to differential interactions between the different isomers of the chromophore and the protein.