Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo.

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.

Could hand-eye laterality profiles affect sport performance? A systematic review.

Background: Laterality effects on sports performance have been a field of interest for the sports sciences, especially in asymmetrical sports, which require the preferential use of one side of the body. Some sports in particular involve the visual system and ocular laterality, due to the need to clearly focus on a dynamic object (ball, opponent, projectile, etc.). The relationship between manual and ocular laterality results in two perceptual-motor profiles, one where the dominant hand and eye are ipsilateral (uncrossed hand-eye laterality profile, UC-HELP), and the other where they are contralateral (crossed hand-eye laterality profile, C-HELP). Methodology: A systematic review of the literature was carried out to determine the prevalence of hand-eye laterality profiles in the different sports modalities and their relationship with psychological factors and sports performance. Searches of PsycInfo, Medline, Scopus and grey literature identified 14 studies (2,759 participants) regarding hand-eye laterality in sports that met the eligibility criteria. Results: Previous studies have estimated that between 10-30% of the general population exhibit a C-HELP, and 70-90% have an UC-HELP. The results of the reviewed studies indicate that in some sports the percentage of C-HELP is higher in regular and high-level athletes than in the normal population: golf (52.55%), soccer (53%), tennis (42%) and team sports (50.7%). In target sports (archery and shooting) athletes with an UC-HELP seem to have an advantage given the significant concentration of this profile in the highest performing populations (82.3%). In basketball, cricket and golf, the literature reviewed also reported biomechanical differences in the execution of some techniques between the two profiles. We did not find any study in our review that related hand-eye laterality with cognitive, tactical, or psychological aspects of athletes. Conclusions: These results should be taken with great caution due to the potential bias linked to the methodologies used in the investigations, the heterogeneity in the assessment of hand-eye laterality, the few studies available on the subject and the indirect nature of many of the observed relationships between performance and laterality. For further investigation, we propose a standardized terminology and protocol of hand-eye laterality assessment in sports. The advancement in knowledge about hand-eye laterality profiles, along with the study of the relationship with psychological or tactical-sports patterns, can contribute to more effective development plans for athletes and can be a complement to talent detection.

On the Computational Design of Azobenzene-Based Multi-State Photoswitches.

In order to theoretically design multi-state photoswitches with specific properties, an exhaustive computational study is first carried out for an azobenzene dimer that has been recently synthesized and experimentally studied. This study allows for a full comprehension of the factors that govern the photoactivated isomerization processes of these molecules so to provide a conceptual/computational protocol that can be applied to generic multi-state photoswitches. From this knowledge a new dimer with a similar chemical design is designed and also fully characterized. Our theoretical calculations predict that the new dimer proposed is one step further in the quest for a double photoswitch, where the four metastable isomers could be selectively interconverted through the use of different irradiation sequences.

Predicting the Electronic Absorption Band Shape of Azobenzene Photoswitches.

Simulations based on molecular dynamics coupled to excitation energy calculations were used to generate simulated absorption spectra for a family of halide derivatives of azobenzene, a family of photoswitch molecules with a weak absorption band around 400-600 nm and potential uses in living tissue. This is a case where using the conventional approach in theoretical spectroscopy (estimation of absorption maxima based on the vertical transition from the potential energy minimum on the ground electronic state) does not provide valid results that explain how the observed band shape extends towards the low energy region of the spectrum. The method affords a reasonable description of the main features of the low-energy UV-Vis spectra of these compounds. A bathochromic trend was detected linked to the size of the halide atom. Analysis of the excitation reveals a correlation between the energy of the molecular orbital where excitation starts and the energy of the highest occupied atomic orbital of the free halide atom. This was put to the test with a new brominated compound with good results. The energy level of the highest occupied orbital on the free halide was identified as a key factor that strongly affects the energy gap in the photoswitch. This opens the way for the design of bathochromically shifted variants of the photoswitch with possible applications.

[Social determinants of the incidence of Covid-19 in Barcelona: a preliminary ecological study using public data.] / Determinantes sociales de la incidencia de la Covid-19 en Barcelona: un estudio ecológico preliminar usando datos públicos.

OBJECTIVE: Social determinants and health inequalities have a huge impact on health of populations. It is important to study their role in the management of the Covid-19 epidemic, especially in cities, as certain variables like the number of tests and the access to health system cannot be assumed as equal. The aim of this work was to determine the relation of social determinants in the incidence of Covid-19 in the city of Barcelona. METHODS: An observational retrospective ecological study was performed, with the neighbourhood as the population unit, based on data of cumulative incidence published at May 14th, 2020 by the Public Health Agency of Barcelona. Covid-19 incidence disparities depending on the income of the neighbourhoods, the Pearson linear correlation of the variables selected (age, sex, net density, immigrants, comorbidities, smokers, Body Mass Index [BMI] and Available Income per Family Index [AIFI]) with the incidence and the correlation with a multivariant Generalized Linear Model (GLM) were estimated. RESULTS: It was found that neighbourhoods belonging to the lowest quintile of income had a 42% more incidence than those belonging to the highest quintile: 942 cases per 100,000 inhabitants versus 545 per 100,000 inhabitants of the highest quintile. The Pearson correlation was statistically significative between the incidence of Covid-19 and the percentage of population over 75 (r=0.487), the percentage of immigration of the neighbourhood and the origin of the immigrants (r=-0.257), the AIFI (r=-0.462), the percentage of smokers (r=0.243) and the percentage of people with BMI over 25 (r=0.483). The GLM showed that the most correlated variables with the incidence are the percentage of people over 75 (Z-score=0.258), the percentage of people from Maghreb (Z-score=-0.206) and Latin America (Z-score=0.19) and the percentage of people with BMI over 25 (Z-score=0.334). The results of the GLM were significative. CONCLUSIONS: Social determinants are correlated with the modification of the incidence of Covid-19 in the neighbourhoods of Barcelona, with special relevance of the prevalence of BMI over 25 and the percentage of immigrants and its origin.

OBJETIVO: Los determinantes sociales tienen un gran impacto en la salud de las poblaciones. Es relevante estudiar su papel en la gestión de la epidemia de la Covid-19, especialmente en las ciudades, pues ciertas variables como el número de tests realizados o la disponibilidad de recursos sanitarios no se pueden asumir por igual. El objetivo de este trabajo fue estimar la relación de los determinantes sociales en la incidencia de la Covid-19 en Barcelona. METODOS: Se realizó un estudio ecológico, observacional retrospectivo, con el barrio como unidad de población, basado en los datos publicados a fecha de 14 de mayo de 2020 sobre incidencia acumulada de Covid-19 confirmada por PCR. Se estimó la diferencia de incidencia de la Covid-19 en función de la renta de los barrios, la correlación lineal de Pearson de las distintas variables seleccionadas (edad, sexo, densidad neta, inmigrantes, comorbilidades, tabaquismo, Índice de Masa Corporal [IMC] e Índice de Renta Familiar Disponible [IRFD]) con la incidencia acumulada y se llevó a cabo un análisis multivariante mediante un Modelo Lineal Generalizado (GLM). RESULTADOS: Los barrios del quintil de menor renta presentaban un 42% más de incidencia que aquellos del quintil con más renta: 942 casos por cada 100.000 habitantes frente a los 545 casos por cada 100.000 habitantes. La correlación de Pearson se mostró estadísticamente significativa entre la incidencia de la Covid-19 y el porcentaje de población mayor de 75 años (r=0,487), el porcentaje de inmigrantes (r=-0,257) y el origen de dichos inmigrantes, el IRFD (r=-0,462), el porcentaje de fumadores (r=0,243) y de personas con un IMC mayor de 25 (r=0,483). En GLM las variables que más correlación tenían con la incidencia entre barrios eran el porcentaje de población mayor de 75 años (Z-score=0,258), el porcentaje de inmigrantes latinoamericanos (Z-score=0,19) y magrebíes (Z-score=-0,206), y el porcentaje de personas con IMC>25 (Z-score=0,334). Los resultados del GLM fueron estadísticamente significativos. CONCLUSIONES: Los determinantes sociales se correlacionan con una modificación de la incidencia de la Covid-19 en los barrios de Barcelona, con especial relevancia de la prevalencia de IMC>25 y del porcentaje de inmigrantes y de su origen.

Determinantes sociales de la incidencia de la Covid-19 en Barcelona: un estudio ecológico preliminar usando datos públicos / Social determinants of the incidence of Covid-19 in Barcelona: a preliminary ecological study using public data

OBJETIVO: Los determinantes sociales tienen un gran impacto en la salud de las poblaciones. Es relevante estudiar su papel en la gestión de la epidemia de la Covid-19, especialmente en las ciudades, pues ciertas variables como el número de tests realizados o la disponibilidad de recursos sanitarios no se pueden asumir por igual. El objetivo de este trabajo fue estimar la relación de los determinantes sociales en la incidencia de la Covid-19 en Barcelona. MÉTODOS: Se realizó un estudio ecológico, observacional retrospectivo, con el barrio como unidad de población, basado en los datos publicados a fecha de 14 de mayo de 2020 sobre incidencia acumulada de Covid-19 confirmada por PCR. Se estimó la diferencia de incidencia de la Covid-19 en función de la renta de los barrios, la correlación lineal de Pearson de las distintas variables seleccionadas (edad, sexo, densidad neta, inmigrantes, comorbilidades, tabaquismo, Índice de Masa Corporal [IMC] e Índice de Renta Familiar Disponible [IRFD]) con la incidencia acumulada y se llevó a cabo un análisis multivariante mediante un Modelo Lineal Generalizado (GLM). RESULTADOS: Los barrios del quintil de menor renta presentaban un 42% más de incidencia que aquellos del quintil con más renta: 942 casos por cada 100.000 habitantes frente a los 545 casos por cada 100.000 habitantes. La correlación de Pearson se mostró estadísticamente significativa entre la incidencia de la Covid-19 y el porcentaje de población mayor de 75 años (r=0,487), el porcentaje de inmigrantes (r=-0,257) y el origen de dichos inmigrantes, el IRFD (r=-0,462), el porcentaje de fumadores (r=0,243) y de personas con un IMC mayor de 25 (r=0,483). En GLM las variables que más correlación tenían con la incidencia entre barrios eran el porcentaje de población mayor de 75 años (Z-score=0,258), el porcentaje de inmigrantes latinoamericanos (Z-score=0,19) y magrebíes (Z-score=-0,206), y el porcentaje de personas con IMC>25 (Z-score=0,334). Los resultados del GLM fueron estadísticamente significativos. CONCLUSIONES: Los determinantes sociales se correlacionan con una modificación de la incidencia de la Covid-19 en los barrios de Barcelona, con especial relevancia de la prevalencia de IMC>25 y del porcentaje de inmigrantes y de su origen

OBJECTIVE: Social determinants and health inequalities have a huge impact on health of populations. It is important to study their role in the management of the Covid-19 epidemic, especially in cities, as certain variables like the number of tests and the access to health system cannot be assumed as equal. The aim of this work was to determine the relation of social determinants in the incidence of Covid-19 in the city of Barcelona. METHODS: An observational retrospective ecological study was performed, with the neighbourhood as the population unit, based on data of cumulative incidence published at May 14th, 2020 by the Public Health Agency of Barcelona. Covid-19 incidence disparities depending on the income of the neighbourhoods, the Pearson linear correlation of the variables selected (age, sex, net density, immigrants, comorbidities, smokers, Body Mass Index [BMI] and Available Income per Family Index [AIFI]) with the incidence and the correlation with a multivariant Generalized Linear Model (GLM) were estimated. RESULTS: It was found that neighbourhoods belonging to the lowest quintile of income had a 42% more incidence than those belonging to the highest quintile: 942 cases per 100,000 inhabitants versus 545 per 100,000 inhabitants of the highest quintile. The Pearson correlation was statistically significative between the incidence of Covid-19 and the percentage of population over 75 (r=0.487), the percentage of immigration of the neighbourhood and the origin of the immigrants (r=-0.257), the AIFI (r=-0.462), the percentage of smokers (r=0.243) and the percentage of people with BMI over 25 (r=0.483). The GLM showed that the most correlated variables with the incidence are the percentage of people over 75 (Z-score=0.258), the percentage of people from Maghreb (Z-score=-0.206) and Latin America (Z-score=0.19) and the percentage of people with BMI over 25 (Z-score=0.334). The results of the GLM were significative. CONCLUSIONS: Social determinants are correlated with the modification of the incidence of Covid-19 in the neighbourhoods of Barcelona, with special relevance of the prevalence of BMI over 25 and the percentage of immigrants and its origin

Deciphering the grounds of the suitability of acylhydrazones as efficient photoswitches.

Many efforts are currently being devoted to designing molecular photoswitches with specific properties. In this sense, a recent publication [D. J. van Dijken et al., J. Am. Chem. Soc., 2015, 137, 14982-14991] has synthesized and analyzed the photochromic properties of a large set of acylhydrazones (ACHs), a relatively unexploited class of potential photoswitches with two stable E and Z isomers. This study has revealed a very diverse and complex pattern of the absorption/emission properties of ACHs depending on the substituents attached to the ACH motif. In this work, high level theoretical calculations are performed on a representative set of the experimentally studied ACHs in order to analyze, at the molecular level, the reasons behind the different photochemistries experimentally observed. This systematic study allows for the classification of the full set of ACHs into just four categories. The two more common groups display a small, either positive or negative, shift of the maximum wavelength of absorption between the E and Z isomers. Less common, but far more interesting from a practical point of view, are the compounds that show a large (>100 nm) Stokes shift. This behavior may arise from two different situations. The most common one implies the possibility of an intramolecular proton transfer in the excited electronic state of the less stable Z isomer. The less likely scenario would also involve a loss of the azidic proton through an intermolecular proton transfer that would take place with the aid of the solvent.

Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation.

Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions.

Experimental and theoretical insights into the influence of electronic density on proton-transfer reactions.

We report on the excited-state behavior of proton-transfer phenanthroimidazole derivatives, such as HPPI and NMHPPI, in solutions using steady-state and femto- to nanosecond time-resolved fluorescence spectroscopies. Experimental observations are supported by theoretical calculations (TDDFT). In dichloromethane (DCM) and acetonitrile (ACN), two different paths are found for the excited-state intramolecular proton-transfer (ESIPT) reactions following two different channels. A fast and direct channel (ESIPT1) in 1-2.5 ps and a slower one (ESIPT2) in 12-15 ps, the latter being more influenced by the solvent viscosity (30 ps for HPPI and 20 ps for NMHPPI in triacetin (TAC) solutions). The slowing down of the ESIPT2 reaction is explained in terms of an intramolecular charge transfer (ICT) reaction coupled to a twisting motion to reach a more suitable conformation of the involved parts in the proton-transfer motion. The absence of OH/OD exchange effects in the ultrafast and slow proton-transfer dynamics suggests that the ESIPT reactions, which involve intramolecular and solvent coordinates, do not occur via tunneling. These results reveal new insights into the photobehavior of proton-transfer dyes, which might help in designing photosensors or lighting devices.

Ultrafast action chemistry in slow motion: atomistic description of the excitation and fluorescence processes in an archetypal fluorescent protein.

We report quantum mechanical/molecular mechanical non-adiabatic molecular dynamics simulations on the electronically excited state of green fluorescent protein mutant S65T/H148D. We examine the driving force of the ultrafast (τ < 50 fs) excited-state proton transfer unleashed by absorption in the A band at 415 nm and propose an atomistic description of the two dynamical regimes experimentally observed [Stoner Ma et al., J. Am. Chem. Soc., 2008, 130, 1227]. These regimes are explained in terms of two sets of successive dynamical events: first the proton transfers quickly from the chromophore to the acceptor Asp148. Thereafter, on a slower time scale, there are geometrical changes in the cavity of the chromophore that involve the distance between the chromophore and Asp148, the planarity of the excited-state chromophore, and the distance between the chromophore and Tyr145. We find two different non-radiative relaxation channels that are operative for structures in the reactant region and that can explain the mismatch between the decay of the emission of A* and the rise of the emission of I*, as well as the temperature dependence of the non-radiative decay rate.

Spectroscopy and dynamics of dehydrobenzo[12]annulene derivatives possessing peripheral carboxyphenyl groups: theory and experiment.

In this work, we report on the results of theoretical and experimental studies of a series of dehydrobenzoannulene (DBA) derivatives (Nu-T12 [5,6,11,12,17,18-hexadehydrotribenzo[a,e,i]cyclododecene], T12-COOMe [5,6,11,12,17,18-hexadehydro-2,3,8,9,14,15-hexakis(4-methoxycarbonylphenyl)tribenzo[a,e,i]cyclododecene] and T12-COOH [5,6,11,12,17,18-hexadehydro-2,3,8,9,14,15-hexakis(4-carboxyphenyl)tribenzo[a,e,i]cyclododecene]) in N,N'-dimethylformamide (DMF) solutions. The theoretical and experimental findings show that the S0 → S1 transition of these molecules is forbidden. Time-resolved spectroscopy measurements determined a lifetime of ∼100 ps of the transition from the first electronical excited (S1) state. These molecules also emit through charge transfer (CT) species, with lifetimes of ∼1 and ∼4.5 ns. In addition to this, Nu-T12 and T12-COOMe in DMF solutions exhibit an emission from their triplet state in 35 and 24.5 ns, respectively. However, T12-COOH strongly interacts through H-bonds with DMF molecules, leading to the formation of new species having a proton-transferred character, whose emission spectrum is red-shifted and its lifetime from the S1 state is ∼25 ns. Using nanosecond (ns) flash photolysis, we also observed the presence of non-emissive triplet states, in addition to the emissive ones. The theoretical calculations suggest that this non-radiative triplet state originates from a CT structure of the emissive triplet one. The new findings presented here elucidate the photobehaviour of three DBA derivatives of relevance to crystalline Hydrogen-Bonded Organic Framework (HOF) materials. The photophysical data provide a strong basis to explore and to better understand the photodynamics of HOF crystals.

Chromophore interactions leading to different absorption spectra in mNeptune1 and mCardinal red fluorescent proteins.

Extensive MD simulations combined with QM/MM calculations have been performed on mNeptune1 and mCardinal red fluorescent proteins to establish the reasons behind the red shift of the excitation wavelength of mCardinal with respect to mNeptune1. In both cases, it is seen that Arg197 stabilizes the chromophore but cannot be described as stabilizing preferentially the excited state because of the anchor point of the interaction. The interactions of the linking bonds to the α-helix of both proteins to the chromophore have been analyzed. It has been found that, besides the presence of a strategically placed residue Gln41 in mCardinal, solvation water molecules play an active role in the energetics of the stabilization of the excited state, which is preferentially stabilized in the case of mCardinal in contrast to mNeptune1.

The Quest for Photoswitches Activated by Near-Infrared Light: A Theoretical Study of the Photochemistry of BF2 -Coordinated Azo Derivatives.

Recently synthesized BF2 -coordinated azo derivatives have been proposed as photoswitches that operate in the optical window (λ=600-1200 nm) for use in bioimaging applications. Herein, we have theoretically analyzed these compounds and modified some substituents to analyze which properties of the molecule govern its photochemistry. Our results compare rather well with the available experimental data, so our methodology, based on density functional theory (DFT) calculations for the ground electronic state and time-dependent-DFT for the first excited electronic state, is validated. Through systematic modification of different substituents of the parent system, we designed compounds that are predicted to operate fully within the optical window. We also analyzed several molecules for which the cis isomer is the more stable isomer, a quite unusual result for azobenzene derivatives that is a much coveted property for some applications of these photoactive molecules in pharmacology. Our results also provide insight into other properties relevant for photoswitches, such as the thermal stability of the less stable isomer and the magnitude of the gap between the wavelengths of the radiation that activates each isomerization process, which must be as large as possible to improve the yield of each photoisomerization. From a more general perspective, our results may provide a step towards the rational design of new photoswitches that fulfill a set of desired characteristics.

Molecular modelling of the pH influence in the geometry and the absorbance spectrum of near-infrared TagRFP675 fluorescent protein.

Classical molecular dynamics (MD) simulations are carried out for the recently developed TagRFP675 fluorescent protein (FP), which is specifically designed to fully absorb and emit in the near infrared (NIR) region of the electromagnetic spectrum. Since the X-ray data of TagRFP675 reveal that the chromophore exists in both the cis and trans configuration and it can also be neutral (protonated) or anionic (deprotonated) depending on the pH of the media, a total of 8 molecular dynamic simulations have been run to simulate all the possible states of the chromophore. Time-dependent DFT (TDDFT) single point calculations are performed at selected points along the simulation to theoretically mimic the absorption spectrum of the protein. Our simulations compare well (within the expected error of the computational method) with the experimental results. Our theoretical procedure allows for an analysis of the molecular orbitals involved in the lowest energy electronic excitations of the chromophore and, more interestingly, for a full analysis of the H-bond interactions between the chromophore and its surrounding residues and solvent (water) molecules. This study does not support the hypothesis, exclusively based on the analysis of X-ray data, that the isomerization of nearby residues provokes the rearrangement of the hydrogen bonds in the chromophore's immediate environment leading to the observed red shift of the absorption bands at higher pHs. Instead, we attribute this shift mainly to the superposition of bands of the neutral and anionic chromophores that are expected to coexist at almost the full range of pHs experimentally analyzed. An additional factor that could contribute to this shift is the experimentally observed increase of the cis configuration of the chromophore at higher pHs.

Transient low-barrier hydrogen bond in the photoactive state of green fluorescent protein.

In this paper, we have analyzed the feasibility of spontaneous proton transfer in GFP at the Franck-Condon region directly after photoexcitation. Computation of a sizeable portion of the potential energy surface at the Franck-Condon region of A the structure shows the process of proton transfer to be unfavorable by 3 kcal mol(-1) in S1 if no further structural relaxation is permitted. The ground vibrational state is found to lie above the potential energy barrier of the proton transfer in both S0 and S1. Expectation values of the geometry reveal that the proton shared between the chromophore and W22, and the proton shared between Ser205 and Glu222 are very close to the center of the respective hydrogen bonds, giving support to the claim that the first transient intermediate detected after photoexcitation (I0*) has characteristics similar to those of a low-barrier hydrogen bond [Di Donato et al., Phys. Chem. Chem. Phys., 2012, 13, 16295]. A quantum dynamical calculation of the evolution in the excited state shows an even larger probability of finding those two protons close to the center compared to in the ground state, but no formation of the proton-transferred product is observed. A QM/MM photoactive state geometry optimization, initiated using a configuration obtained by taking the A minimum and moving the protons to the product side, yields a minimum energy structure with the protons transferred and in which the His148 residue is substantially closer to the now anionic chromophore. These results indicate that: (1) proton transfer is not possible if structural relaxation of the surroundings of the chromophore is prevented; (2) protons H1 and H3 especially are found very close to the point halfway between the donor and acceptor after photoexcitation when the zero-point energy is considered; (3) a geometrical parameter exists (the His148-Cro distance) under which the structure with the protons transferred is not a minimum, and that, if included, should lead to the fluorescing I* structure. The existence of an oscillating stationary state between the reactants and products of the triple proton transfer reaction can explain the dual emission reported for the I0* intermediate of wtGFP.

Direct observation of breaking of the intramolecular H-bond, and slowing down of the proton motion and tuning its mechanism in an HBO derivative.

We report on spectroscopic and photodynamical behaviours of 5-amino-2-(2'-hydroxyphenyl)benzoxazole (5A-HBO) in different solutions. The dye undergoes an ultrafast ICT reaction (<50 fs) (comparable to that observed for its methylated derivative, 5A-MBO), in agreement with the results of TD-DFT theoretical calculations (gas phase). Depending on the used solvent, the ICT reaction can be followed by a reversible/irreversible excited-state intramolecular proton transfer (ESIPT) reaction or by breaking of the intramolecular hydrogen bond (IHB). 5A-HBO in n-heptane solution exhibits an irreversible and slow (20 ps) ESIPT reaction, while that of the parent compound, HBO, takes place in less than 150 fs. Compared to excited HBO behaviour, theoretical calculations on 5A-HBO suggest a higher energy barrier (∼4 kcal mol(-1)) between the relaxed enol and keto tautomers, in addition to a less stabilization of the latter, which is in agreement with experiments in n-heptane. On the other hand, in dichloromethane, after the ICT reaction a subsequent and reversible proton motion occurs in an extraordinary slower regime (ns-time scale). No isotopic effect (OH/OD exchange) was observed in this solvent reflecting that the reversible ESIPT reaction evolves along the IHB and solvent coordinates. Using tetrahydrofurane and acetonitrile, we observed a breaking of the IHB due to specific intermolecular interactions with solvent molecules. This leads to the formation of open-enol forms, which undergo an ICT reaction as it occurs in 5A-MBO. These results bring new findings in the coupled ICT and ESIPT reactions. The photobehaviour of this new dye remarkably changes with the solvent nature, opening up the window for further research and possible applications in sensing polarity or H-bonding of media similar to that of the biological ones.

Theoretical Computer-Aided Mutagenic Study on the Triple Green Fluorescent Protein Mutant S65T/H148D/Y145F.

Green fluorescent protein (GFP) mutant S65T/H148D has been proposed to host a photocycle that involves an excited-state proton transfer between the chromophore (Cro) and the Asp148 residue and takes place in less than 50 fs without a measurable kinetic isotope effect. It has been suggested that the interaction between the unsuspected Tyr145 residue and the chromophore is needed for the ultrafast sub-50 fs rise in fluorescence. To verify this, we have performed a computer-aided mutagenic study to introduce the additional mutation Y145F, which eliminates this interaction. By means of QM/MM molecular dynamics simulations and time-dependent density functional theory studies, we have assessed the importance of the Cro-Tyr145 interaction and the solvation of Asp148 and shown that in the triple mutant S65T/H148D/Y145F a significant loss in the ultrafast rise of the Stokes-shifted fluorescence should be expected.

Unveiling how an archetypal fluorescent protein operates: theoretical perspective on the ultrafast excited state dynamics of GFP variant S65T/H148D.

Green fluorescent protein variant S65T/H148D has been reported to host a photocycle involving the photoinduced proton transfer reaction between the chromophore and residue Asp148 under 50 fs and without a measurable kinetic isotope effect, and experimental evidence is suggestive of the existence of a highly delocalized proton between these residues. The blinding speed at which this biological system undergoes proton transfer has been ascribed to the extreme increase of acidity of the GFP chromophore in the electronic excited state where proton transfer takes place. This work strives to present a coherent, complete, and balanced description of the dynamics of this specific variant of GFP in which it will be shown that this increase of acidity is insufficient to explain the behavior observed. This study tracks the behavior of this photosystem to the delicate interplay between structure and dynamics shown in the presence of solvent. In this way, it has been found that the dynamics of this protein intertwines its structure with the intervening solvent to give rise to effectively degenerate situations in what concerns the reactants and products of the proton transfer reaction in ground and, most importantly, photoexcited state, in terms of potential energy profiles associated with the proton migration. Under these conditions, proton transfer can occur in accordance with the experimental data available. This set of characteristics is possibly common to a host of other proton transfer based fluorescent proteins, and helps promoting GFP S65T/H148D to a case of archetypal significance. Thus, our results can be useful to understand the way many fluorescent proteins work and, more generally, the molecular basis for proton transfer reactions in proteins.

New insights into the structure-spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment.

The green fluorescent protein (GFP) variant S65T/H148D recovers the A-band fluorescence lost in the single mutant S65T, and it has been established that Asp148 is the alternate proton acceptor for the excited state proton transfer (ESPT). This mutant has been widely studied and presents unique spectroscopic properties, such as an ultrafast rise in the fluorescence (<50 fs). Also it exhibits a red-shift of the A absorption band of 20 nm with respect to wt-GFP's. The double mutant E222Q/H148D presents a very similar behaviour, at least within the experimental data available (which is scarcer than those of S65T/H148D). By means of dynamic theoretical studies we have been able to (1) reproduce and thoroughly analyse the red-shifted absorption spectra of both mutants and (2) predict the structure that the variant E222Q/H148D (for which there is no X-ray-resolved structure available) most probably adopts in water at room temperature. Our results deepen the understanding of the way GFP variants work and give some new insights into the rational design of fluorescent proteins and biological photosystems in general.

Are there really low-barrier hydrogen bonds in proteins? The case of photoactive yellow protein.

For a long time, low-barrier hydrogen bonds (LBHBs) have been proposed to exist in many enzymes and to play an important role in their catalytic function, but the proof of their existence has been elusive. The transient formation of an LBHB in a protein system has been detected for the first time using neutron diffraction techniques on a photoactive yellow protein (PYP) crystal in a study published in 2009 (Yamaguchi, S.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 440-444). However, very recent theoretical studies based on electronic structure calculations and NMR resonance experiments on PYP in solution (Saito, K.; et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 167-172) strongly indicate that there is not such an LBHB. By means of electronic structure calculations combined with the solution of the nuclear Schrödinger equation, we analyze here under which conditions an LBHB can exist in PYP, thus leading to a more reasonable and conciliating understanding of the above-mentioned studies.