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Green‐to‐red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super‐resolution imaging. A unique Kaede‐type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady‐state and time‐resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green‐to‐red photoconversion. However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA‐A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π–π stacking interaction between the chromophore phenolate (P‐)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of ~60–67 ps are attributed to P‐ring twist as the onset for photoswitching in LEA (major) and LEA‐A69T (minor) with photoconversion capability, different from ~16/29 ps in correlation with the Gln62/His62 side‐chain twist in ALL‐GFP/ALL‐Q62H, indicative of the light‐induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA‐A69T capable of positive photoswitching was revealed by time‐resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green‐to‐red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances. [ABSTRACT FROM AUTHOR]

The mechanism of chromophore formation in green fluorescent protein (GFP) involves its post-translational self-modification process which yields an intrinsic fluorophore constructed from an internal main-chain cross-link. A mechanistic model of GFP involving conformational pre-organization, electrophilic and base catalysts, and production of hydrogen peroxide upon protein oxidation is developed to better understand the process.

Research has been conducted on the green fluorescent protein chromophore. The examination of the Raman spectra that has been obtained using 752 nm excitation is discussed.

The spectral and structural response of green fluorescent protein mutants of the jellyfish Aequorea victoria to pH variations is investigated. To this end, the high-resolution crystal structures of the S65T variant are analyzed. Results show changes in the hydrogen bond pattern with the chromophore, indicating that the pH sensitivity arises from protonation of the chromophore phenolate. pH titrations of the variants suggest that the chromophore pK(sub a) can be modulated over a wide range from 6 to 8, resulting in pH determination from pH 5 to pH 9.

The mechanisms of synchronous and asynchronous C-H and C-X bond cleavage were analyzed during the ER2 halide elimination of beta-haloethanols. Analysis of the ionic elimination reactions of galatose oxidase (GOase) indicated the presence of a significant primary deuterium effect on the rate of inactivation. The rate of inactivation was also affected by the leaving group which also induced the formation of a long-lived anion intermediate. Furthermore, the mechanism-based inactivation for GOase involved two electron transfer reactions from the substrate to the enzyme.

An experiment is conducted to determine the crystal structure of a blue emission variant of the jellyfish 'Aequorea victoria' green fluorescent protein using molecular replacement. X-ray diffraction data and samples of blue fluorescent protein are used for the study. Results show that the chromophore in the intact protein is electrically neutral. In addition, fluorescence transmission is not identified with excited-state photon transfer.

Galactose oxidase (GOase) is a fungal enzyme that catalyzes the oxidation of primary alcohols, producing hydrogen peroxide and aldehydes. It contains two one-electron redox centers: a mononuclear copper center, and a tyrosine center that is cross-linked to cysteine. Analysis of the active site of this enzyme by replacing the primary alcohol group with substrate analogs incorporating a primary thiol group provided evidence linking thiols with inactivation of the enzyme. It was suggested that thiols may be potential active-site-directed redox inactivators.

Stepwise or concerted binding mechanisms in the galactose oxidase active site are investigated using molecular mechanics calculations of radical carbon-hydrogen bond cleavage as the rate-determining step in hexopyranose turnover. Results indicate that the active site of galactose oxidase is evolved enough to catalyze the radical oxidation of alcohols to aldehydes with either stepwise or concerted binding mechanisms.

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light‐induced processes. We implemented time‐resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP‐like protein LEA, involving semi‐trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual‐illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge‐H bending motions enables rational design of functional proteins. With the improved H‐bonding network and structural motions, the photoexcited chromophore could increase the photoswitching‐aided photoconversion while reducing trapped species. [ABSTRACT FROM AUTHOR]

A series of main chain redox and [beta]-elimination reactions mediated by light and [O.sub.2], yielding a red-emitting chromophore is described. The studies have described yellow fluorescence arising from modifications of DsRed-type intermediates and photoactivated oxidative redding phenomenon along with the two major classes of red fluorescent proteins.

Isotope-enriched protein bearing tyrosine residues deuterated at the beta carbon was prepared to determine the kinetic isotope effects (KIEs) on the GFP self-processing reaction. The data obtained are consistent with Arg96, which serves as the general base that catalyzes proton abstraction from the Tyr66-derived beta carbon.

The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), which is a variant of enhanced cyan fluorescent protein (ECFP), is determined. The results have shown that the high-resolution X-ray structure of Cerulean GFP is consistent with a single ground state conformation for the chromophore and its protein environment, although the broad absorbance doublet is persisting in the crystal.

The kinetics of hydrogen peroxide evolution during in vitro green fluorescent protein (GFP) maturation is investigated and it is found that the H2O2 coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The molecular mass of an oxidized intermediate is characterized and it is found that, in air-saturated aqueous solutions, a dehydrogenated species is generated.

A study demonstrates that the initially colorless Y66L variant, designed to trap chromophore precursor states, is oxidatively modified to generate yellow chromophore that absorbs at 412 and 374 nm. The data suggest that hydration-dehydration equilibrium exists in the cyclic form of the peptide, and that dehydration is favored upon extensive conjugation with the modified side chain.

Structural details of the crystal tetrameric yellow-fluorescent protein zFP538 from the button polyp Zoanthus species and a green-emitting mutant (K66M) are presented. The atomic models of these proteins are specified and its role in chemistry of chromophore maturation is mentioned.

A novel mechanism for green fluorescent protein (GFP) chromophore formation is proposed, on the basis of the x-ray structure and mass spectrometry results. The replacement of an aromatic with an aliphatic residue (Y66L) in GFP has a profound effect on the efficiency of dehydration of the cyclic intermediate.

Steady state absorption, fluorescence, and raman spectroscopies are used to investigate the response of wild-type green fluorescent protein (GFP) to UV and visible light. It is reported that the rate of light-driven decarboxylation reaction strongly depends on the excitation wavelength, decreasing in the order 254 nm > 280 nm > 476 nm.

Summary: Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO2) and ribulose‐1,5‐bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to pterin‐4α‐ carbinolamine dehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD‐like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana. [ABSTRACT FROM AUTHOR]

Photoconvertible fluorescent proteins (pcFPs) constitute a large group of fluorescent proteins related to green fluorescent protein (GFP) that, when exposed to blue light, bear the capability of irreversibly switching their emission color from green to red. Not surprisingly, this fascinating class of FPs has found numerous applications, in particular for the visualization of biological processes. A detailed understanding of the photoconversion mechanism appears indispensable in the design of improved variants for applications such as super-resolution imaging. In this article, recent work is reviewed that involves using pcFPs as a model system for studying protein dynamics. Evidence has been provided that the evolution of pcFPs from a green ancestor involved the natural selection for altered dynamical features of the beta-barrel fold. It appears that photoconversion may be the outcome of a long-range positional shift of a fold-anchoring region. A relatively stiff, rigid element appears to have migrated away from the chromophore-bearing section to the opposite edge of the barrel, thereby endowing pcFPs with increased active site flexibility while keeping the fold intact. In this way, the stage was set for the coupling of light absorption with subsequent chemical transformations. The emerging mechanistic model suggests that highly specific dynamic motions are linked to key chemical steps, preparing the system for a concerted deprotonation and β-elimination reaction that enlarges the chromophore's π-conjugation to generate red color. [ABSTRACT FROM AUTHOR]