Biology subjects

De Zitter, E.

Publications and source records attributed to De Zitter, E..

8 recordsLinked to original sources

bioRxiv2026

Structural basis of the two-photon photoactivation mechanism of orange carotenoid protein

Cyanobacteria have produced Earths oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity- dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCPO) generates a metastable intermediate (OCP1h{nu}) that either relaxes thermally or, upon absorption of a second photon within [~]1 s, converts to the active photoprotective state (OCPR). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP1h{nu}, establish its functional relevance and capture structural snapshots along the OCPO[->]OCP1h{nu} and OCP1h{nu}[->]OCPR photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.

biochemistry↗

bioRxiv2025

Integrated structural dynamics uncover new modes of B12 photoreceptor activation

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The molecular mechanism of B12 photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B12 photoreceptor CarH4,5 from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co-C bond triggers a pathway of structural changes that propagate throughout CarH from the B12 chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B12 enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B12-dependent photoreceptors for future optogenetic applications.

biophysics↗

bioRxiv2023

Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4{degrees}C and a few days at 37{degrees}C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.

biophysics↗

bioRxiv2022

Oligomerization processes limit photoactivation and recovery of the Orange Carotenoid Protein.

The Orange Carotenoid Protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection, by quenching of the excess of light harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy quenching activity. Here, we probed photo-induced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation, a numbed-state forms, which remains in a non-photoexcitable structural state for at least [~]0.5 {micro}s after absorption of a first photon. Significance StatementThe orange carotenoid protein (OCP) is a photoactivatable protein involved in cyanobacterial photoprotection. Upon photoactivation, OCP becomes able to quench the excess of energy uptaken by the light-harvesting antennae, thereby evading damage to the cells. It remains unclear, however, what is the exact OCP photoactivation mechanism, and whether or not oligomerization partakes in the regulation of the OCP function. Here, we investigated these issues by combining static and time-resolved (TR) X-ray scattering and optical spectroscopy. Our results show that OCP oligomerizes in both the dark-adapted inactive and light-adapted active states, suggesting a functional role for oligomerization. TR scattering data furthermore reveal that the first large-scale conformational changes associated with OCP photoactivation take place on the {micro}s time scale.

biophysics↗

bioRxiv2022

Xtrapol8: automatic elucidation of low-occupancy intermediate-states in crystallographic studies

Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, hindering structure determination by conventional methods. To automatically extract such structures, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of hitherto unavailable methods; (iii) calculates various types of extrapolated structure factors while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures.

biophysics↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

bioRxiv2020

Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

biophysics↗

bioRxiv2018

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort.

biophysics↗