How electron tunneling and uphill excitation energy transfer support photochemistry in Halomicronema hongdechloris.

Halomicronema hongdechloris, the first cyanobacterium reported to produce the red-shifted chlorophyll f (Chl f) upon acclimation to far-red light, demonstrates remarkable adaptability to diverse light conditions. The photosystem II (PS II) of this organism undergoes reversible changes in its Chl f content, ranging from practically zero under white-light culture conditions to a Chl f: Chl a ratio of up to 1:8 when exposed to far-red light (FRL) of 720-730 nm for several days. Our ps time- and wavelength-resolved fluorescence data obtained after excitation of living H. hongdechloris cells indicate that the Soret band of a far-red (FR) chlorophyll involved in charge separation absorbs around 470 nm. At 10 K, the fluorescence decay at 715-720 nm is still fast with a time constant of 165 ps indicating an efficient electron tunneling process. There is efficient excitation energy transfer (EET) from 715-720 nm to 745 nm with the latter resulting from FR Chl f, which mainly functions as light-harvesting pigment upon adaptation to FRL. From there, excitation energy reaches the primary donor in the reaction center of PS II with an energetic uphill EET mechanism inducing charge transfer. The fluorescence data are well explained with a secondary donor PD1 represented by a red-shifted Chl a molecule with characteristic fluorescence around 715 nm and a more red-shifted FR Chl f with fluorescence around 725 nm as primary donor at the ChlD1 or PD2 position.

Effect of Molecular Dynamics and Internal Water Contact on the Photophysical Properties of Red pH-Sensitive Proteins.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the 1S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Thermoresponsive swelling of photoacoustic single-chain nanoparticles.

NIR-fluorescent LCST-type single-chain nanoparticles (SCNPs) change their photophysical behaviour upon heating, caused by depletion of water from the swollen SCNP interiors. This thermoresponsive effect leads to a fluctuating photoacoustic (PA) signal which can be used as a contrast mechanism for PA imaging.

Light-Induced Conformational Flexibility of the Orange Carotenoid Protein Studied by Quasielastic Neutron Scattering with In Situ Illumination.

The orange carotenoid protein plays a vital role in the photoprotection of cyanobacteria and exhibits a significant structural change upon photoactivation. A rarely considered aspect is the importance of internal protein dynamics in facilitating the structural transition to the active state. In this study, we use quasielastic neutron scattering under (in situ) blue light illumination for the first time to directly probe the protein dynamics of the orange carotenoid protein in the dark-adapted and active states. This shows that the localized internal dynamics of amino acid residues is significantly enhanced upon photoactivation. This is attributed to the photoinduced structural changes exposing larger areas of the protein surface to the solvent, thus resulting in a higher degree of motional freedom. However, the flexibility of the W288A mutant assumed to mimic the active state structure is found to be different, thus highlighting the importance of in situ experiments.

Tuning the Internal Compartmentation of Single-Chain Nanoparticles as Fluorescent Contrast Agents.

Controlling the internal structures of single-chain nanoparticles (SCNPs) is an important factor for their targeted chemical design and synthesis, especially in view of nanosized compartments presenting different local environments as a main feature to control functionality. We here design SCNPs bearing near-infrared fluorescent dyes embedded in hydrophobic compartments for use as contrast agents in pump-probe photoacoustic (PA) imaging, displaying improved properties by the location of the dye in the hydrophobic particle core. Compartment formation is controlled via single-chain collapse and subsequent crosslinking of an amphiphilic polymer using external crosslinkers in reaction media of adjustable polarity. Different SCNPs with hydrodynamic diameters of 6-12 nm bearing adjustable label densities are synthesized. It is found that the specific conditions for single-chain collapse have a major impact on the formation of the desired core-shell structure, in turn adjusting the internal nanocompartments together with the formation of excitonic dye couples, which in turn increase their fluorescence lifetime and PA signal generation. SCNPs with the dye molecules accumulate at the core also show a nonlinear PA response as a function of pulse energy-a property that can be exploited as a contrast mechanism in molecular PA tomography.

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of "molecular surgery" to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical ß-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual ß-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level ("molecular surgery"), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of ß-barrel fluorescent proteins.

Coordination-Induced Band Gap Reduction in a Metal-Organic Framework.

Herein, we report on the synthesis of a microporous, three-dimensional phosphonate metal-organic framework (MOF) with the composition Cu3 (H5 -MTPPA)2 ⋅ 2 NMP (H8 -MTPPA=methane tetra-p-phenylphosphonic acid and NMP=N-methyl-2-pyrrolidone). This MOF, termed TUB1, has a unique one-dimensional inorganic building unit composed of square planar and distorted trigonal bipyramidal copper atoms. It possesses a (calculated) BET surface area of 766.2 m2 /g after removal of the solvents from the voids. The Tauc plot for TUB1 yields indirect and direct band gaps of 2.4 eV and 2.7 eV, respectively. DFT calculations reveal the existence of two spin-dependent gaps of 2.60 eV and 0.48 eV for the alpha and beta spins, respectively, with the lowest unoccupied crystal orbital for both gaps predominantly residing on the square planar copper atoms. The projected density of states suggests that the presence of the square planar copper atoms reduces the overall band gap of TUB1, as the beta-gap for the trigonal bipyramidal copper atoms is 3.72 eV.

Arylphosphonate-Tethered Porphyrins: Fluorescence Silencing Speaks a Metal Language in Living Enterocytes*.

We report the application of a highly versatile and engineerable novel sensor platform to monitor biologically significant and toxic metal ions in live human Caco-2 enterocytes. The extended conjugation between the fluorescent porphyrin core and metal ions through aromatic phenylphosphonic acid tethers generates a unique turn off and turn on fluorescence and, in addition, shifts in absorption and emission spectra for zinc, cobalt, cadmium and mercury. The reported fluorescent probes p-H8 TPPA and m-H8 TPPA can monitor a wide range of metal ion concentrations via fluorescence titration and also via fluorescence decay curves. Cu- and Zn-induced turn off fluorescence can be differentially reversed by the addition of common chelators. Both p-H8 TPPA and m-H8 TPPA readily pass the mammalian cellular membrane due to their amphipathic character as confirmed by confocal microscopic imaging of living enterocytes.

Courses Based on iGEM/BIOMOD Competitions Are the Ideal Format for Research-Based Learning of Xenobiology.

Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on "iGEM-Synthetic Biology" offered at the Technische Universität Berlin as an example.

Fluorescent and Water Dispersible Single-Chain Nanoparticles: Core-Shell Structured Compartmentation.

Single-chain nanoparticles (SCNPs) are highly versatile structures resembling proteins, able to function as catalysts or biomedical delivery systems. Based on their synthesis by single-chain collapse into nanoparticular systems, their internal structure is complex, resulting in nanosized domains preformed during the crosslinking process. In this study we present proof of such nanocompartments within SCNPs via a combination of electron paramagnetic resonance (EPR) and fluorescence spectroscopy. A novel strategy to encapsulate labels within these water dispersible SCNPs with hydrodynamic radii of ≈5 nm is presented, based on amphiphilic polymers with additional covalently bound labels, attached via the copper catalyzed azide/alkyne "click" reaction (CuAAC). A detailed profile of the interior of the SCNPs and the labels' microenvironment was obtained via electron paramagnetic resonance (EPR) experiments, followed by an assessment of their photophysical properties.

Phosphonate Metal-Organic Frameworks: A Novel Family of Semiconductors.

Herein, the first semiconducting and magnetic phosphonate metal-organic framework (MOF), TUB75, is reported, which contains a 1D inorganic building unit composed of a zigzag chain of corner-sharing copper dimers. The solid-state UV-vis spectrum of TUB75 reveals the existence of a narrow bandgap of 1.4 eV, which agrees well with the density functional theory (DFT)-calculated bandgap of 1.77 eV. Single-crystal conductivity measurements for different orientations of the individual crystals yield a range of conductances from 10-3 to 103 S m-1 at room temperature, pointing to the directional nature of the electrical conductivity in TUB75. Magnetization measurements show that TUB75 is composed of antiferromagnetically coupled copper dimer chains. Due to their rich structural chemistry and exceptionally high thermal/chemical stabilities, phosphonate MOFs like TUB75 may open new vistas in engineerable electrodes for supercapacitors.

Melanoidin formed from fructosylalanine contains more alanine than melanoidin formed from d-glucose with L-alanine.

In this study the elemental compositions of melanoidin formed at 160 °C from d-glucose (Glc) and l-alanine (Ala) as well as from fructosylalanine - the corresponding Amadori rearrangement product - were compared. Specific chemical bonds were probed by FTIR spectroscopy. This approach tackles the different chemical pathways for melanoidin formation via the Amadori rearrangement in contrast to the reaction from Glc/Ala. Melanoidins formed from fructosylalanine contain about twice as much nitrogen and therefore amino acid as compared to melanoidin from Glc/Ala and exhibit higher absorption in the UV/Vis. Consequently, melanoidins formed from Glc/Ala contain more sugar degradation products with lower absorption due to a smaller size of the conjugated double bond network.

Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet-visible (UV-vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C. For the O600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O600. This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering.

Orange carotenoid proteins (OCPs) are photoswitchable macromolecules playing an important role in nonphotochemical quenching of excess energy in cyanobacterial light harvesting. Upon absorption of a blue photon (450-500 nm), OCPs undergo a structural change from the ground state OCPO to the active state OCPR, but high-resolution structures of the active state OCPR are not yet available. Here, we use small-angle scattering methods combined with simulation tools to determine low-resolution structures of the active state at low protein concentrations via two approaches: first, directly by in situ illumination of wild-type OCP achieving a turnover to the active state of >90% and second, by using the mutant OCPW288A anticipated to mimic the active state structure. Data fits assuming the shape of an ellipsoid yield three ellipsoidal radii of about 9, 29, and 51 ± 1 Å, in the case of the ground state OCPO. In the active state, however, the molecule becomes somewhat narrower with the two smaller radii being 9 and only 19 ± 3 Å, while the third dimension of the ellipsoid is significantly elongated to 85-92 ± 5 Å. Reconstitutions of the active state structure corroborate that OCPR is significantly elongated compared to the ground state OCPO and characterized by a separation of the N-terminal and C-terminal domains with unfolded N-terminal extension. By direct comparison of small-angle scattering data, we directly show that the mutant OCPW288A can be used as a structural analogue of the active state OCPR. The small-angle experiments are repeated for OCPO and the mutant OCPW288A at high protein concentrations of 50-65 mg/mL required for neutron spectroscopy investigating the molecular dynamics of OCP (see accompanying paper). The results reveal that the OCPO and OCPW288A samples for dynamics experiments are preferentially dimeric and widely resemble the structures of the ground and active states of OCP, respectively. This enables us to properly characterize the molecular dynamics of both states of OCP in the accompanying paper.

Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein. Part II: Quasielastic Neutron Scattering.

Orange carotenoid proteins (OCPs), which are protecting cyanobacterial light-harvesting antennae from photodamage, undergo a pronounced structural change upon light absorption. In addition, the active state is anticipated to boost a significantly higher molecular flexibility similar to a "molten globule" state. Here, we used quasielastic neutron scattering to directly characterize the vibrational and conformational molecular dynamics of OCP in its ground and active states, respectively, on the picosecond time scale. At a temperature of 100 K, we observe mainly (vibronic) inelastic features with peak energies at 5 and 6 meV (40 and 48 cm-1, respectively). At physiological temperatures, however, two (Lorentzian) quasielastic components represent localized protein motions, that is, stochastic structural fluctuations of protein side chains between various conformational substates of the protein. Global diffusion of OCP is not observed on the given time scale. The slower Lorentzian component is affected by illumination and can be well-characterized by a jump-diffusion model. While the jump diffusion constant D is (2.82 ± 0.01) × 10-5 cm2/s at 300 K in the ground state, it is increased by ∼20% to (3.48 ± 0.01) × 10-5 cm2/s in the active state, revealing a strong enhancement of molecular mobility. The increased mobility is also reflected in the average atomic mean square displacement ⟨u2⟩; we determine a ⟨u2⟩ of 1.47 ± 0.05 Å in the ground state, but 1.86 ± 0.05 Å in the active state (at 300 K). This effect is assigned to two factors: (i) the elongated structure of the active state with two widely separated protein domains is characterized by a larger number of surface residues with a concomitantly higher degree of motional freedom and (ii) a larger number of hydration water molecules bound at the surface of the protein. We thus conclude that the active state of the orange carotenoid protein displays an enhanced conformational dynamics. The higher degree of flexibility may provide additional channels for nonradiative decay so that harmful excess energy can be more efficiently converted to heat.

Vinylene-Linked Covalent Organic Frameworks by Base-Catalyzed Aldol Condensation.

Two 2D covalent organic frameworks (COFs) linked by vinylene (-CH=CH-) groups (V-COF-1 and V-COF-2) are synthesized by exploiting the electron deficient nature of the aromatic s-triazine unit of C3 -symmetric 2,4,6-trimethyl-s-triazine (TMT). The acidic terminal methyl hydrogens of TMT can easily be abstracted by a base, resulting in a stabilized carbanion, which further undergoes aldol condensation with multitopic aryl aldehydes to be reticulated into extended crystalline frameworks (V-COFs). Both V-COF-1 (with terepthalaldehyde (TA)) and V-COF-2 (with 1,3,5-tris(p-formylphenyl)benzene (TFPB)) are polycrystalline and exhibit permanent porosity and BET surface areas of 1341 m2 g-1 and 627 m2 g-1 , respectively. Owing to the close proximity (3.52 Å) of the pre-organized vinylene linkages within adjacent 2D layers stacked in eclipsed fashion, [2+2] photo-cycloadditon in V-COF-1 formed covalent crosslinks between the COF layers.

PCA-based identification and differentiation of FTIR data from model melanoidins with specific molecular compositions.

Melanoidins formed from different carbohydrates, such as d-glucose, d-fructose, and d-xylose, and their typical degradation products, such as hydroxymethylfurfural, furfural, glyoxal, and methylglyoxal, with l-alanine were analyzed with Fourier transform infrared spectroscopy (FTIR). Characteristic infrared bands were identified representing spectral differences between the investigated melanoidin species due to their different molecular compositions. With the help of principal components analysis (PCA) the IR data allowed for a fast discrimination between the different model melanoidins. From this study it is inferred that the intensity and relative absorption wavelength of CO single versus CO double bonds are characteristic features of the investigated melanoidins. Melanoidins formed from carbohydrates exhibit less carbonyl functions in comparison to melanoidins from the degradation products, the situation is opposite for the CO bond. The amount of CO is additionally correlated with a higher absorption at 420 nm indicating that strong colored melanoidins contain more carbonyl functions.

Photosynthesis supported by a chlorophyll f-dependent, entropy-driven uphill energy transfer in Halomicronema hongdechloris cells adapted to far-red light.

The phototrophic cyanobacterium Halomicronema hongdechloris shows far-red light-induced accumulation of chlorophyll (Chl) f, but the involvement of the pigment in photosynthetic energy harvesting by photosystem (PS) II is controversially discussed. While H. hongdechloris contains negligible amounts of Chl f in white-light culture conditions, the ratio of Chl f to Chl a is reversibly changed up to 1:8 under illumination with far-red light (720-730 nm). We performed UV-Vis absorption spectroscopy, time-integrated and time-resolved fluorescence spectroscopy for the calculation of decay-associated spectra (DAS) to determine excitation energy transfer (EET) processes between photosynthetic pigments in intact H. hongdechloris filaments. In cells grown under white light, highly efficient EET occurs from phycobilisomes (PBSs) to Chl a with an apparent time constant of about 100 ps. Charge separation occurs with a typical apparent time constant of 200-300 ps from Chl a. After 3-4 days of growth under far-red light, robust Chl f content was observed in H. hongdechloris and EET from PBSs reached Chl f efficiently within 200 ps. It is proposed based on mathematical modeling by rate equation systems for EET between the PBSs and PSII and subsequent electron transfer (ET) that charge separation occurs from Chl a and excitation energy is funneled from Chl f to Chl a via an energetically uphill EET driven by entropy, which is effective because the number of Chl a molecules coupled to Chl f is at least eight- to tenfold larger than the corresponding number of Chl f molecules. The long lifetime of Chl f molecules in contact to a tenfold larger pool of Chl a molecules allows Chl f to act as an intermediate energy storage level, from which the Gibbs free energy difference between Chl f and Chl a can be overcome by taking advantage from the favorable ratio of degeneracy coefficients, which formally represents a significant entropy gain in the Eyring formulation of the Arrhenius law. Direct evidence for energetically uphill EET and charge separation in PSII upon excitation of Chl f via anti-Stokes fluorescence in far-red light-adapted H. hongdechloris cells was obtained: Excitation by 720 nm laser light resulted in robust Chl a fluorescence at 680 nm that was distinctly temperature-dependent and, notably, increased upon DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) treatment in far-red light-adapted cells. Thus, rather than serving as an excitation energy trap, Chl f in far-red light-adapted H. hongdechloris cells is directly contributing to oxygenic photosynthesis at PSII.

Molecular insights into antibiotic resistance - how a binding protein traps albicidin.

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs.

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence.

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.