Excited-state dynamics and fluorescence lifetime of cryogenically cooled green fluorescent protein chromophore anions.

Time-resolved action spectroscopy together with a fs-pump probe scheme is used in an electrostatic ion-storage ring to address lifetimes of specific vibrational levels in electronically excited states. Here we specifically consider the excited-state lifetime of cryogenically cooled green fluorescent protein (GFP) chromophore anions which is systematically measured across the S0-S1 spectral region (450-482 nm). A long lifetime of 5.2 ± 0.3 ns is measured at the S0-S1 band origin. When exciting higher vibrational levels in S1, the lifetime changes dramatically. It decreases by more than two orders of magnitude in a narrow energy region ∼250 cm-1 (31 meV) above the 0-0 transition. This is attributed to the opening of internal conversion over an excited-state energy barrier. The applied experimental technique provides a new way to uncover even small energy barriers, which are crucial for excited-state dynamics.

Stress/depression across the COVID-19 pandemic in Denmark.

BACKGROUND: Global estimates suggest strained mental health during the first year of the COVID-19 pandemic, but the lack of nationally representative and longitudinal data with clinically validated measures limits knowledge longer into the pandemic. METHODS: Data from 10 rounds of nationally representative surveys from Denmark tracked trends in risk of stress/depression from just before the first lockdown and through to April 2022. We focused on age groups and men and women in different living arrangements and controlled for seasonality in mental health that could otherwise be spuriously related to pandemic intensity. RESULTS: Prior to first lockdown, we observed a "parent gap", which closed with the first lockdown. Instead, a gender gap materialized, with women experiencing higher risks than men-and higher than levels predating first lockdown. Older respondents (+ 70 years) experienced increasing risks of stress/depression early in the pandemic, while all other groups experienced decreases. But longer into the pandemic, risks increased for all age groups and reached (and sometimes exceeded) levels from before first lockdown. CONCLUSION: Denmark had low infection rates throughout most of the pandemic, low mortality rates across the entire pandemic, and offered financial aid packages to curb financial strains. Despite this circumstance, initial improvements to mental health during the first lockdown in Denmark were short-lived. Two years of pandemic societal restrictions correspond with deteriorating mental health, as well as a change from a parenthood gap in mental health before first lockdown to a gender gap two years into the pandemic.

Light Driven Ultrafast Bioinspired Molecular Motors: Steering and Accelerating Photoisomerization Dynamics of Retinal.

Photoisomerization of retinal protonated Schiff base in microbial and animal rhodopsins are strikingly ultrafast and highly specific. Both protein environments provide conditions for fine-tuning the photochemistry of their chromophores. Here, by combining time-resolved action absorption spectroscopy and high-level electronic structure theory, we show that similar control can be gained in a synthetically engineered retinal chromophore. By locking the dimethylated retinal Schiff base at the C11═C12 double bond in its trans configuration (L-RSB), the excited-state decay is rendered from a slow picosecond to an ultrafast subpicosecond regime in the gas phase. Steric hindrance and pretwisting of L-RSB are found to be important for a significant reduction in the excited-state energy barriers, where isomerization of the locked chromophore proceeds along C9═C10 rather than the preferred C11═C12 isomerization path. Remarkably, the accelerated excited-state dynamics also becomes steered. We show that L-RSB is capable of unidirectional 360° rotation from all-trans to 9-cis and from 9-cis to all-trans in only two distinct steps induced by consecutive absorption of two 600 nm photons. This opens a way for the rational design of red-light-driven ultrafast molecular rotary motors based on locked retinal chromophores.

Controlling Light-Induced Proton Transfer from the GFP Chromophore.

The front cover artwork is provided by the groups of Assoc. Prof. Anastasia V. Bochenkova (Lomonosov Moscow State University) and Prof. Lars H. Andersen (Aarhus University). The image shows the quantum nature of wavelength-dependent excited-state proton transfer in gas-phase H-bonded complexes of the GFP chromophore with an anionic proton acceptor. Read the full text of the Article at 10.1002/cphc.202100068.

Controlling Light-Induced Proton Transfer from the GFP Chromophore.

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I- and deprotonated trichloroacetic acid (TCA- ). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S1 , revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S1 , depending on the quantum-density distribution upon vibrational excitation. We also show that the S1 potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.

Tuning fast excited-state decay by ligand attachment in isolated chlorophyll a.

Excited-state dynamics plays a key role for light harvesting and energy transport in photosynthetic proteins but it is nontrivial to separate the intrinsic photophysics of the light-absorbers (chlorophylls) from interactions with the protein matrix. Here we study chlorophyll a (4-coordinate complex) and axially ligated chlorophyll a (5-coordinate complex) isolated in vacuo applying mass spectrometry to shed light on the intrinsic dynamics in the absence of nearby chlorophylls, carotenoids, amino acids, and water molecules. The 4-coordinate complexes are tagged by quaternary ammonium ions while the charge is provided by a formate ligand in the case of 5-coordinate complexes. Regardless of excitation to the Soret band or the Q band, a fast ps decay is observed, which is ascribed to the decay of the lowest excited singlet state either by intersystem crossing (ISC) to nearby triplet states or by excited-state relaxation on the excited-state potential-energy surface. The lifetime of the first excited state is 15 ps with Mg2+ at the chlorophyll center, but only 1.7 ps when formate is attached to Mg2+. When the Soret band is excited, an initial sup-ps relaxation is observed which is ascribed to fast internal conversion to the first excited state. With respect to ISC, two factors seem to play a role for the reduced lifetime of the formate-chlorophyll complex: (i) The Mg ion is pulled out of the porphyrin plane thus reducing the symmetry of the chromophore, and (ii) the first excited state (Q band) and T3 are tuned almost into resonance by the ligand, which increases the singlet-triplet mixing.

Spectroscopy and photoisomerization of protonated Schiff-base retinal derivatives in vacuo.

The protonated Schiff-base retinal acts as the chromophore in bacteriorhodopsin as well as in rhodopsin. In both cases, photoexcitation initializes fast isomerization which eventually results in storage of chemical energy or signaling. The details of the photophysics for this important chromophore is still not fully understood. In this study, action-absorption spectra and photoisomerization dynamics of three retinal derivatives are measured in the gas phase and compared to that of the protonated Schiff-base retinal. The retinal derivatives include C9C10trans-locked, C13C14trans-locked and a retinal derivative without the ß-ionone ring. The spectroscopy as well as the isomerization speed of the chromophores are altered significantly as a consequence of the steric constraints.

Risk of stress/depression and functional impairment in Denmark immediately following a COVID-19 shutdown.

BACKGROUND: Existing estimates of the impact of the COVID-19 burden on mental wellbeing come from countries with high mortality rates. This study therefore aimed to investigate the impact of the first COVID-19 lockdown (March-April 2020) on risk for stress/depression and functional impairment in a representative sample of adult individuals in Denmark, which had lower infection rates, and whether the impact of lockdown was heterogeneous across living situation. METHODS: Using a representative, randomly drawn sample from the complete Danish adult population interviewed in March 2 to April 13, 2020 (n = 2836) and again in July 2020 (n = 1526, 54% retention rate), we study how the imposed lockdown announced March 11 following the onset of the first Danish wave of COVID-19 infections affected mental wellbeing. We use the World Health Organization Five Well-being Index (WHO-5) and the Work and Social Adjustment Scale (WSAS) to capture risk for stress/depression (WHO-5 < 50) and functional impairment (WSAS > 10). Using covariate adjusted ordinary least squares linear probability models and exploiting variation in the timing of responses occurring just before and just after the introduction of lockdown, we compare respondents before lockdown to respondents that answered during lockdown, as well as to answers in re-interviews in July. RESULTS: In our fully controlled models, we find reduced depressive symptoms among adults immediately after the shutdown, concentrated in adults with children living at home (-.089, p < .01 (from pre lockdown baseline .273)). Measures of functional impairment also declined immediately after the March shutdown among adults with children living at home (-.066, p < .05 (from pre lockdown baseline .150)). Impairment intensified for the entire sample between March and July (+.199, p < .001 (from pre lockdown baseline .248)), but depressive symptoms remained at lower rate in July (-.033, p < .05 (from pre lockdown baseline .332). CONCLUSIONS: Findings in Denmark indicate that living with children at home may have, in the short term, buffered the potential mental health sequelae of the COVID-19 shutdown.

Color tuning of chlorophyll a and b pigments revealed from gas-phase spectroscopy.

Chlorophyll (Chl) pigments are responsible for vital mechanisms in photosynthetic proteins: light harvesting, energy transfer and charge separation. A complex interplay between the Chl molecule and its microenvironment determines its transition energy. Interactions such as excitonic coupling with one or more pigments (Chls or carotenoids), axial ligation to the magnesium center, or electrostatic interactions between Chl and nearby amino-acid residues all influence the photophysical properties. Here we use time-resolved photodissociation action spectroscopy to determine transition energies of Chla/b complexes in vacuo to directly compare the impact of a negatively charged axial ligand (formate) to that of exciton coupling between two Chls. Experiments carried out at the electrostatic ion storage ring ELISA allow dissociation to be sampled on hundreds of milliseconds time scale. Absorption-band maxima of Chla-formate complexes are found at 433 ± 4 nm/2.86 ± 0.03 eV (Soret band) and in the region 654-675 nm/1.84-1.90 eV (Q band) and those of Chla dimers tagged by a quaternary ammonium ion at 419 ± 5 nm/2.96 ± 0.04 eV (Soret band) and 647 nm/1.92 eV (Q band). The axial ligand strongly affects the Chla transition energies causing redshifts of 0.21 eV of the Soret band and 0.04-0.1 eV of the Q band compared to Chla tagged by a quaternary ammonium. Slightly smaller shifts were found in case of Chlb. The redshifts are approximately twice that induced by excitonic coupling between two Chlas, also tagged by a quaternary ammonium ion. Axial ligation brings the absorption by isolated Chls very close to that of photosynthetic proteins.

Intrinsic Photophysics of Light-harvesting Charge-tagged Chlorophyll a and b Pigments.

Chlorophylls a and b (Chla/b) are responsible for light-harvesting by photosynthetic proteins in plants. They display broad absorption in the visible region with multiple bands, due to the asymmetry of the macrocycle and strong vibronic coupling. Their photophysics relies on the microenvironment, with regard to transition energies as well as quenching of triplet states. Here, we firmly establish the splitting of the Q and Soret bands into x- and y- polarized bands for the isolated molecules in vacuo, and resolve vibronic features. Storage-ring experiments reveal that dissociation of photoexcited charge-tagged complexes occurs over several milliseconds, but with two different time constants. A fast decay is ascribed to dissociation after internal conversion and a slow decay to the population of a triplet state that acts as a bottleneck. Support for the latter is provided by pump-probe experiments, where a second laser pulse probes the long-lived triplet state.

Action-spectroscopy studies of positively charge-tagged azobenzene in solution and in the gas-phase.

The absorption of a positively charge-tagged azobenzene molecule is studied in the gas-phase by measuring photoinduced fragmentation of ions as a function of time. This technique provides information on prompt as well as delayed fragmentation, and a single dissociation channel after one-photon absorption is identified. The spectra in solution, as well as in the gas-phase, show a weak S0 → S1, a strong S0 → S2, and a broad absorption band in the UV regime. The bands are assigned through time dependent density functional theory calculations. The ratio of the various absorption bands depends on the trans to cis isomerization fraction and may be tuned by light irradiation. Gas-phase absorption spectra are presented and discussed in terms of trans and cis isomers.

The Effect of an Electric Field on the Spectroscopic Properties of the Isolated Green Fluorescent Protein Chromophore Anion.

Here we uncover the direct effect of a high electric field on the absorption by the Green Fluorescent Protein chromophore anion isolated in vacuo based on gas-phase action spectroscopy. Betaine is a strong molecular dipole that creates an electric field of ∼70 MV/cm when attached to the ion at the phenolate oxygen, more than half the actual field from the protein matrix and pointing in the same direction. Nevertheless, the shift in absorption is limited (0.08 eV), supporting earlier conclusions, but subject to much debate, that the protein is rather innocent in perturbing the transition energy. The betaine complexes are readily made by electrospray ionization and in contrast to the bare ions, they dissociate after one-photon absorption. Also, electron detachment is not an open channel complicating the bare ion case. As steric constraints are absent in vacuo, the possibility of turning on fluorescence by an electric field can be tested from experiments on complexes with betaine.

The UV-visible action-absorption spectrum of all-trans and 11-cis protonated Schiff base retinal in the gas phase.

The UV-visible absorption of retinal in its protonated Schiff-base form is studied in the gas phase. In particular, transitions to highly-excited electronic states, Sn, in the all-trans and 11-cis forms are considered, and several new states are discovered. Their positions and strengths are compared to state of the art quantum calculations. The location of these states are particularly important when new fs pump-probe experiments are designed to investigate the fast excited-state dynamics of retinal chromophores.

Elucidation of the intrinsic optical properties of hydrogen-bonded and protonated flavin chromophores by photodissociation action spectroscopy.

A model system of the flavin chromophore was synthesized and investigated for its intrinsic optical properties by gas phase action spectroscopy using an ion storage ring. An ammonium group was anchored to this flavin chromophore to allow its transfer to the gas phase by electrospray ionization and for studying the influence of hydrogen bonding and a nearby positive charge. According to calculations one of the hydrogen atoms of the ammonium group favorably forms an intramolecular ionic hydrogen bond to one of the oxygen atoms of the flavin chromophore, and this interaction was found to cause a blueshift of the S0 → S1 transition and a redshift of the S0 → S2 transition. For comparison, the S0 → S1 transition shows little solvent dependence (only in regard to the degree of fine structure). In addition, the influence of protonation of the flavin chromophore was elucidated by experimental and theoretical studies of a simple flavin system. While the position of the S0 → S1 absorption was at identical positions in the gas phase for the intramolecularly hydrogen-bonded and protonated flavin systems, the S0 → S2 absorption was further redshifted for the protonated species. This redshift resulting from protonation was also observed in solution.

Origin of the Intrinsic Fluorescence of the Green Fluorescent Protein.

Green fluorescent protein, GFP, has revolutionized biology, due to its use in bioimaging. It is widely accepted that the protein environment makes its chromophore fluoresce, whereas the fluorescence is completely lost when the native chromophore is taken out of GFP. By the use of a new femtosecond pump-probe scheme, based on time-resolved action spectroscopy, we demonstrate that the isolated deprotonated GFP chromophore can be trapped in the first excited state when cooled to 100 K. The trapping is shown to last for 1.2 ns, which is long enough to establish conditions for fluorescence and consistent with calculated trapping barriers in the electronically excited state. Thus, GFP fluorescence is traced back to an intrinsic chromophore property, and by improving excited-state trapping, protein interactions enhance the molecular fluorescence.

Explaining the Consequences of Imprisonment for Union Formation and Dissolution in Denmark.

Crime and subsequent imprisonment reduces men's chances on the marriage market and increases their divorce risk, but existing research, with a few notable exceptions, is silent about the underlying mechanisms driving these effects. This article studies the effect of home confinement under electronic monitoring as a noncustodial alternative to imprisonment on the risk of relationship dissolution and being single, thereby distinguishing between effects of incarceration and of committing crime. We study a policy that expanded the use of electronic monitoring to address nonrandom selection into electronic monitoring instead of in prison. Results from a sample of 4,522 men show that home confinement under electronic monitoring significantly and persistently lowers the risk both of being single and of becoming single during the first five years following conviction. The results show that one of the tools that could promote decarceration trends also secures better relationship outcomes of convicted men.

Decoupling Electronic versus Nuclear Photoresponse of Isolated Green Fluorescent Protein Chromophores Using Short Laser Pulses.

The photophysics of a deprotonated model chromophore for the green fluorescent protein is studied by femtosecond laser pulses in an electrostatic ion-storage ring. The laser-pulse duration is much shorter than the time for internal conversion, and, hence, contributions from sequential multiphoton absorption, typically encountered with ns-laser pulses, are avoided. Following single-photon excitation, the action-absorption maximum is shown to be shifted within the S_{0} to S_{1} band from its origin at about 490 to 450 nm, which is explained by the different photophysics involved in the detected action.

A PYP chromophore acts as a 'photoacid' in an isolated hydrogen bonded complex.

The light-induced response of a neutral Photoactive Yellow Protein chromophore in a hydrogen-bonded complex with a proton acceptor has been studied by dual-detection action absorption spectroscopy and density functional theory. We show that the chromophore is a 'photoacid' and that ultrafast excited-state proton transfer might be operative in an isolated complex.

How Children's Educational Outcomes and Criminality Vary by Duration and Frequency of Paternal Incarceration.

Existing studies of the consequences of paternal incarceration for children treat paternal incarceration as a dichotomous event (a child either experiences paternal incarceration or does not), although effects could accumulate with both the frequency and duration of paternal incarcerations. In this article I use register data on Danish children from birth cohort 1991, some of whom experienced paternal incarceration before age 15, to show how educational outcomes and criminality up to age 20 vary by frequency and total duration of paternal incarceration. The high quality of Danish register data also allows me to distinguish between paternal arrest and paternal incarceration and to show results for the total duration of paternal incarcerations conditioned on frequency of paternal incarceration. Results show that educational outcomes and criminality indeed correlate with duration and frequency of paternal incarceration, indicating that treating paternal incarceration as a dichotomous event blurs important heterogeneity in the consequences of paternal incarceration.