Chromophore Environment Research Articles

Photophysical Behavior of mNeonGreen, an Evolutionary Distant Green Fluorescent Protein

Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion-proteins in model systems and live cells. In particular, molecular brightness depends on the dynamics of short-lived dark states leading to flickering of the fluorescence signal. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from Branchiostoma lanceolatum, in comparison to EGFP and AcGFP from Aequorea species. Fluorescence correlation spectroscopy (FCS) is used to determine flickering and molecular brightness of purified proteins in solutions of different pH. The molecular brightness of mNG is consistently higher as compared to the traditional GFPs, but decreases slightly under acidic and basic conditions. Consistently, an advanced analysis of steady state spectroscopy data resolves two additional chromophore states with reduced quantum yield that build up at low and high pH. Thus, mNG is distinguished from traditional GFPs by a two-step protonation response that reflects a chemically distinct chromophore environment. Despite the more complex pH dependence, mNG represents a superior GFP under a broad range of conditions.

Rational Protein Design via Structure-Energetics-Function Relationships in the Photoactive Yellow Protein (PYP) Model System

Rational protein design uses biophysical principles to inform the generation of new, purpose-built proteins. However, this methodology is hampered by our limited understanding of these biophysical principles relating a protein's structure to its function. In an initial attempt at using structure-energetics-function relationships to rationally design protein function, we recently developed a model that guides qualitative suggestions for engineering light-activated dissociation of split GFPs [1]. We analyzed the potential energy surface of the photodissociation reaction coordinate to identify critical energetics features like barriers and population branching ratios, which can be tuned to increase the photoswitching quantum yield and decrease the fluorescence quantum yield. To develop this structure-energetics-function design methodology further, we are using QM/MM non-adiabatic dynamics studies to predict the effects of mutating key residues in the PYP chromophore environment on observable photoactivated processes. These predictions will then be evaluated by comparisons to spectroscopic measurements.

Chromophore Isomer Stabilization Is Critical to the Efficient Fluorescence of Cyan Fluorescent Proteins.

ECFP, the first usable cyan fluorescent protein (CFP), was obtained by adapting the tyrosine-based chromophore environment in green fluorescent protein to that of a tryptophan-based one. This first-generation CFP was superseded by the popular Cerulean, CyPet, and SCFP3A that were engineered by rational and random mutagenesis, yet the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the third-generation CFP mTurquoise and its successors without an obvious rationale. We show here that the evolution process had unexpectedly remodeled the chromophore environment in second-generation CFPs so they would accommodate a different isomer, whose formation is favored by acidic pH or light irradiation and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters and provide novel insights into the rational evolution of fluorescent proteins with a tryptophan-based chromophore.

Absorption Spectra for Disordered Aggregates of Chromophores Using the Exciton Model.

Optimizing the optical properties of large chromophore aggregates and molecular solids for applications in photovoltaics and nonlinear optics is an outstanding challenge. It requires efficient and reliable computational models that must be validated against accurate theoretical methods. We show that linear absorption spectra calculated using the molecular exciton model agree well with spectra calculated using time-dependent density functional theory and configuration interaction singles for aggregates of strongly polar chromophores. Similar agreement is obtained for a hybrid functional (B3LYP), a long-range corrected hybrid functional (ωB97X), and configuration interaction singles. Accounting for the electrostatic environment of individual chromophores in the parametrization of the exciton model with the inclusion of atomic point charges significantly improves the agreement of the resulting spectra with those calculated using all-electron methods; different charge definitions (Mulliken and ChelpG) yield similar results. We find that there is a size-dependent error in the exciton model compared with all-electron methods, but for aggregates with more than six chromophores, the errors change slowly with the number of chromophores in the aggregate. Our results validate the use of the molecular exciton model for predicting the absorption spectra of bulk molecular solids; its formalism also allows straightforward extension to calculations of nonlinear optical response.

Simulation of the UV/Visible Absorption Spectra of Fluorescent Protein Chromophore Models

AbstractTime‐dependent density functional theory has been used to simulate the UV/Vis absorption spectra of fluorescent protein model chromophores with particular focus on the vibronic structure of the lowest‐energy absorption band as a structural fingerprint. Combining the B3LYP‐35 XC functional with non‐equilibrium solvation and the gradient approach provides a vibronic structure close to that recorded experimentally (when comparisons with experiment are possible). This study focuses on the chromophore present in green fluorescent proteins (FP1), in its neutral and deprotonated forms as well as for both cis (Z) and trans (E) conformations. The method was then employed to simulate the spectra of structures for which experimental spectra are not available or not well resolved. The effect of extending the conjugation of the chromophore, by adding unsaturated moieties such as in the chromophore of DsRed (FP2), was considered. Overall, among these three investigated structural modifications (neutral versus anionic, smaller versus larger chromophore, and cis versus trans conformations), absorption spectra can only distinguish unambiguously between the neutral and anionic forms. An extension of the π‐conjugation length of the chromophore is, as would be expected, reflected in a bathochromic shift of the absorption band maximum, but modifications of the chromophore environment in proteins can also induce shifts of the absorption maximum, and they are difficult to distinguish from each other.

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry’s fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.

Blue fluorescent protein derived from the mutated purple chromoprotein isolated from the sea anemone Stichodactyla haddoni.

Chromoproteins, especially far-red fluorescent proteins with long stokes shift, are good sources for engineering biological research tools. However, chromoproteins have not been used for developing fluorescent proteins with short emission wavelength. Therefore, we herein report the development of a blue fluorescent protein, termed shBFP, which is derived from a purple chromoprotein isolated from the sea anemone Stichodacyla haddoni (shCP) after shCP was simultaneously mutated on E63L and Y64L. The shBFP chromophore is composed of Leu-Leu-Gly, which introduced a maximum excitation and emission wavelength at 401 nm and 458 nm, respectively, and a quantum yield of 0.79. Interestingly, the N158S and L173I double mutations of shBFP conducted in the chromophore environment further shifted the maximum excitation to 375 nm, and elevated the quantum yield to 0.84. Thus, shBFP, which is based on the Leu-Leu-Gly chromophore composition, results in higher quantum yields and short wavelength emission. Additionally, we found that the cDNA of shBFP is stably expressed in zebrafish embryos with fidelity, indicating the application of shBFP as a biomarker or selective marker.

Bottom-Up Hierarchical Self-Assembly of Chiral Porphyrins through Coordination and Hydrogen Bonds.

A series of chiral synthetic compounds is reported that shows intricate but specific hierarchical assembly because of varying positions of coordination and hydrogen bonds. The evolution of the aggregates (followed by absorption spectroscopy and temperature-dependent circular dichroism studies in solution) reveal the influence of the proportion of stereogenic centers in the side groups connected to the chromophore ring in their optical activity and the important role of pyridyl groups in the self-assembly of these chiral macrocycles. The optical activity spans 2 orders of magnitude depending on composition and constitution. Two of the aggregates show very high optical activity even though the isolated chromophores barely give a circular dichroism signal. Molecular modeling of the aggregates, starting from the pyridine-zinc(II) porphyrin interaction and working up, and calculation of the circular dichroism signal confirm the origin of this optical activity as the chiral supramolecular organization of the molecules. The aggregates show a broad absorption range, between approximately 390 and 475 nm for the transitions associated with the Soret region alone, that spans wavelengths far more than the isolated chromophore. The supramolecular assemblies of the metalloporphyrins in solution were deposited onto highly oriented pyrolitic graphite in order to study their hierarchy in assembly by atomic force microscopy. Zero and one-dimensional aggregates were observed, and a clear dependence on deposition temperature was shown, indicating that the hierarchical assembly took place largely in solution. Moreover, scanning electron microscopy images of porphyrins and metalloporphyrins precipitated under out-of-equilibrium conditions showed the dependence of the number and position of chiral amide groups in the formation of a fibrillar nanomaterial. The combination of coordination and hydrogen bonding in the complicated assembly of these molecules-where there is a clear hierarchy for zinc(II)-pyridyl interaction followed by hydrogen-bonding between amide groups, and then van der Waals interactions-paves the way for the preparation of molecular materials with multiple chromophore environments.

Introduction of fluorine to change the dielectric environment of nonlinear optical chromophores for improved electro-optic activities

Fluorine was non-conjugatedly linked to the donor of nonlinear optical chromophore to investigate the effect of highly electronegative fluorine on intramolecular charge-transfer and electro-optic activities. 1HNMR spectra and UV–vis absorption were used to investigate that introduction of fluorine change the dielectric environment of intramolecular charge-transfer. Fluorinated chromophore showed more intensified absorption and its absorption properties was more stable to dielectric change in solution. In solid state of guest-host electro-optic polymer films, this dielectric environment stabilization was revealed to attenuate the dipole–dipole interactions of chromophores. Higher electro-optic coefficients (148pm/V) and higher chromophore loading density (30wt%) of fluorinated material chr-2/APC than benchmark material chr-1/APC (116pm/V at 25wt%) was achieved.

Chromophore Deprotonation State Alters the Optical Properties of Blue Chromoprotein.

Chromoproteins (CPs) have unique colors and can be used in biological applications. In this work, a novel blue CP with a maximum absorption peak (λmax) at 608 nm was identified from the carpet anemone Stichodactyla gigantea (sgBP). In vivo expression of sgBP in zebrafish would change the appearance of the fishes to have a blue color, indicating the potential biomarker function. To enhance the color properties, the crystal structure of sgBP at 2.25 Å resolution was determined to allow structure-based protein engineering. Among the mutations conducted in the Gln-Tyr-Gly chromophore and chromophore environment, a S157C mutation shifted the λmax to 604 nm with an extinction coefficient (ε) of 58,029 M-1·cm-1 and darkened the blue color expression. The S157C mutation in the sgBP chromophore environment could affect the color expression by altering the deprotonation state of the phenolic group in the chromophore. Our results provide a structural basis for the blue color enhancement of the biomarker development.

Primary light-induced reaction steps of reversibly photoswitchable fluorescent protein Padron0.9 investigated by femtosecond spectroscopy.

The reversible photoswitching of the photochromic fluorescent protein Padron0.9 involves a cis-trans isomerization of the chromophore. Both isomers are subjected to a protonation equilibrium between a neutral and a deprotonated form. The observed pH dependent absorption spectra require at least two protonating groups in the chromophore environment modulating its proton affinity. Using femtosecond transient absorption spectroscopy, we elucidate the primary reaction steps of selectively excited chromophore species. Employing kinetic and spectral modeling of the time dependent transients, we identify intermediate states and their spectra. Excitation of the deprotonated trans species is followed by excited state relaxation and internal conversion to a hot ground state on a time scale of 1.1-6.5 ps. As the switching yield is very low (Φtrans→cis = 0.0003 ± 0.0001), direct formation of the cis isomer in the time-resolved experiment is not observed. The reverse switching route involves excitation of the neutral cis chromophore. A strong H/D isotope effect reveals the initial reaction step to be an excited state proton transfer with a rate constant of kH = (1.7 ps)(-1) (kD = (8.6 ps)(-1)) competing with internal conversion (kic = (4.5 ps)(-1)). The deprotonated excited cis intermediate relaxes to the well-known long-lived fluorescent species (kr = (24 ps)(-1)). The switching quantum yield is determined to be low as well, Φcis→trans = 0.02 ± 0.01. Excitation of both the neutral and deprotonated cis chromophores is followed by a ground state proton transfer reaction partially re-establishing the disturbed ground state equilibrium within 1.6 ps (deuterated species: 5.6 ps). The incomplete equilibration reveals an inhomogeneous population of deprotonated cis species which equilibrate on different time scales.

Hydrogen Bond Flexibility and Water Dynamics in the Far Red Fluorescent Protein TagRFP675

Next-generation far-red emitting fluorescent proteins (FPs) with enhanced fluorescence quantum yields and high photostability are highly desirable. They would enable deeper tissue penetration and lengthened imaging times given a lower autofluorescence background, lower light scattering, and higher transmission beyond 650 nm. The farthest red emitting FP engineered to date is TagRFP675, which has a 77 nm Stokes shift. The red emission of TagRFP675 is attributed to several H-bonding contacts involving Q41 and S28 at the N-acylimine position with additional H-bonds at the phenoxy end of the chromophore with N143, N158, and R197. We used molecular dynamics (MD) simulations to explore the relationship between flexibility of the chromophore environment and the large Stokes shift in TagRFP675 and related variants mKate and its mutant mKate-M41Q. Both TagRFP675 and mKate-M41Q have large Stokes shift as compared to mKate. Analysis of the hydrogen bonds around the chromophore reveal that in both TagRFP675 and mKate-M41Q have an extended hydrogen bond network connecting Q106-water-F65-Q41-S28 whereas in mKate, this network does not extend beyond F65. The water molecule involved in the hydrogen bond network shows significantly larger flexibility and mobility in TagRFP675 as compared to mKate-M41Q, highlighting the role of the chromophore environment for large bathochromic shifts.

Hydrogen Bond Flexibility Correlates with Stokes Shift in mPlum Variants

Fluorescent proteins have revolutionized molecular biology research and provide a means of tracking subcellular processes with extraordinary spatial and temporal precision. Species with emission beyond 650 nm offer the potential for deeper tissue penetration and lengthened imaging times; however, the origin of their extended Stokes shift is not fully understood. We employed spectrally resolved transient grating spectroscopy and molecular dynamics simulations to investigate the relationship between the flexibility of the chromophore environment and Stokes shift in mPlum. We examined excited state solvation dynamics in a panel of strategic point mutants of residues E16 and I65 proposed to participate in a hydrogen-bonding interaction thought responsible for its red-shifted emission. We observed two characteristic relaxation constants of a few picoseconds and tens of picoseconds that were assigned to survival times of direct and water-mediated hydrogen bonds at the 16-65 position. Moreover, variants of the largest Stokes shift (mPlum, I65V) exhibited significant decay on both time scales, indicating the bathochromic shift correlates with a facile switching between a direct and water-mediated hydrogen bond. This dynamic model underscores the role of environmental flexibility in the mechanism of excited state solvation and provides a template for engineering next-generation red fluorescent proteins.

Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors.

As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutations near the flavin chromophore modulate response kinetics and the effective light responsiveness. To probe for potential, inadvertent effects on receptor activity, we introduced these mutations into the engineered LOV photoreceptor YF1 and determined their impact on light regulation. While several mutations severely impaired the dynamic range of the receptor (e.g., I39V, R63K, and N94A), residue substitutions in a second group were benign with little effect on regulation (e.g., V28T, N37C, and L82I). Electron paramagnetic resonance and absorption spectroscopy identified correlated effects for certain of the latter mutations on chromophore environment and response kinetics in YF1 and the LOV2 domain from Avena sativa phototropin 1. Carefully chosen mutations provide a powerful means to adjust the light-response function of photoreceptors as demanded for diverse applications.

On mechanisms of fluorescence quenching by water

Mechanisms of fluorescence quenching of aromatic chromophores by water are reviewed. The mechanisms include polarity of chromophore environment, proton or electron transfer between the excited chromophore and water. A hypothesis is proposed that the quenching can be a result of chromophore-solvent hydrogen bond breaking in the excited state.

3P246 PYP のβ4-5 loop 領域と発色団環境との関係性の解明(光生物 : 視覚・光受容,ポスター,第52回日本生物物理学会年会(2014年度))

3P246 PYP のβ4-5 loop 領域と発色団環境との関係性の解明(光生物 : 視覚・光受容,ポスター,第52回日本生物物理学会年会(2014年度))

Temperature Dependence of the Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Protein Mplum

We used molecular dynamics (MD) simulations to investigate the dynamics of the hydrogen bonds formed due to protein-chromophore interactions in the far-red fluorescent protein mPlum. Low temperature experiments on mPlum show a reduced Stokes shift compared to room temperature. This suggests that the increased flexibility of the chromophore environment at higher temperatures along with its ability to reorganize after excitation is related to the larger Stokes shift. We performed MD simulations at various temperatures and systematically explored the protein-chromophore hydrogen bond pattern at various temperatures. We also investigated the dynamics of the hydrogen bond formed between residues E16 and I65, which is considered to play an important role in the observed large red shift in the emission spectrum. We quantify the hydrogen bonding pattern as a function of temperature to show how the Stokes shift in mPlum is correlated to the E16-I65 hydrogen bond dynamics.

Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator

As part of an ongoing effort to develop genetically encoded calcium ion (Ca2+) indicators we recently described a new variant, designated CH-GECO2.1, that is a genetic chimera of the red fluorescent protein (FP) mCherry, calmodulin (CaM), and a peptide that binds to Ca2+-bound CaM. In contrast to the closely related Ca2+ indicator R-GECO1, CH-GECO2.1 is characterized by a much higher affinity for Ca2+ and a sensing mechanism that does not involve direct modulation of the chromophore pKa. To probe the structural basis underlying the differences between CH-GECO2.1 and R-GECO1, and to gain a better understanding of the mechanism of CH-GECO2.1, we have constructed, purified, and characterized a large number of variants with strategic amino acid substitutions. This effort led us to identify Gln163 as the key residue involved in the conformational change that transduces the Ca2+ binding event into a change in the chromophore environment. In addition, we demonstrate that many of the substitutions that differentiate CH-GECO2.1 and R-GECO1 have little influence on both the Kd for Ca2+ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles.

Extended Stokes Shift in Fluorescent Proteins: Chromophore–Protein Interactions in a Near-Infrared TagRFP675 Variant

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10–45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis.

The yellow fluorescent protein phiYFPv (λem(max) ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow-orange range (535-555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The `yellow' chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.