Biophysics - Highlights

• Oxygen diffusion pathways in mutated forms of a LOV photoreceptor from Methylobacterium radiotolerans: A molecular dynamics study

Zerlotti R.; Losi A.; Polverini E.; Biomolecular Concepts, 13(1): 164-174 (2022) https://doi.org/10.1515/bmc-2022-0013.

Mr4511 from Methylobacterium radiotolerans is a photoreceptor of the light, oxygen voltage (LOV) family, binding flavin mononucleotide (FMN) as a chromophore.
It exhibits the prototypical LOV photocycle, with the reversible formation of an FMN-Cys71 adduct via fast decay of the FMN triplet state. Mr4511 has high potential as a photosensitiser for singlet oxygen (SO) upon mutation of C71. Mr4511-C71S shows a triplet lifetime (τT) of several hundreds ofmicroseconds, ensuring efficient energy transfer to dioxygen to form SO. In this work, we have explored the potential diffusion pathways for dioxygen within Mr4511 using molecular dynamics (MD) simulations.
The structural model of wild-type (wt) Mr4511 showed a dimeric structure stabilised by a strong leucine zipper at the two C-terminal helical ends. We then
introduced in silico the C71S mutation and analysed transient and persistent oxygen channels. MD simulations indicate that the chromophore binding site is
highly accessible to dioxygen. Mutations that might favour SO generation were designed based on their position with respect to FMN and the oxygen channels.
In particular, the C71S-Y61T and C71S-Y61S variants showed an increased diffusion and persistence of oxygen molecules inside the binding cavity.

• Time‑resolved photoacoustics of channelrhodopsins: early energetics and light‑driven volume changes

Walter M.; Schubert L.; Heberle J.; Schlesinger R.; Losi A.; Photochem Photobiol Sci (2022) https://doi.org/10.1007/s43630-022-00327-8

In biological photoreceptors, the energy stored in early transient species is a key feature to drive the photocycle or a chain of reactions. Time-resolved photoacoustics (PA) can explore the energy landscape of transient species formed within few ns after photoexcitation, as well as volumetric changes (ΔV) of these intermediates with respect to the parental state. In this work, PA identified these important parameters for several channelrhodopsins, namely CaChR1 from Chlamydomonas augustae and CrChR2 from Chlamydomonas reinhardtii and various variants. PA has access to the sub-ns formation of the early photoproduct P1 and to its relaxation, provided that this latter process occurs within a few μs. We found that ΔVP1 for CaChR1 is ca. 12 mL/mol, while it is much smaller for CrChR2 (4.7 mL/mol) and for H. salinarum bacteriorhodopsin (HsBR, ΔVK = 2.8 mL/mol). PA experiments on variants strongly indicate that part of this large ΔVP1 value for CaChR1 is caused by the protonation dynamics of the Schiff base counterion complex involving E169 and D299. PA data further show that the energy level of P1 is higher in CrChR2 (ca. 96 kJ/mol) than in CaChr1 (ca. 46 kJ/mol), comparable to the energy level of the K state of HsBR (60 kJ/mol). Instrumental to gain these molecular values from the raw PA data was the estimation of the quantum yield (Φ) for P1 formation via transient spectroscopy; for both channelrhodopsins, ΦP2 was evaluated as ca. 0.4.

• Mapping the role of aromatic amino acids within a blue-light sensing LOV domain

Ding Y.; Zhao A.Z.; Matysik A.J.; Gartner A.W; Losi A.; Phys. Chem. Chem. Phys., 23: 16767 (2021) https://doi.org/10.1039/d1cp02217b

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical. Tryptophans and tyrosines are held as the main partners as potential electron donors to the flavin excited states. In this work, we explore the relevance of aromatic amino acids in determining the photophysical features of the LOV protein Mr4511 from Methylobacterium radiotolerans by introducing point mutations into the C71S variant that does not form the covalent adduct. By using an array of spectroscopic techniques we measured the fluorescence quantum yields and lifetimes, the triplet yields and lifetimes, and the efficiency of singlet oxygen (SO) formation for eleven Mr4511 variants. Insertion of Trp residues at distances between 0.6 and 1.5 nm from the flavin chromophore results in strong quenching of the flavin excited triplet state and, at the shorter distances even of the singlet excited state. The mutation F130W (ca. 0.6 nm) completely quenches the singlet excited state, preventing triplet formation: in this case, even if the cysteine is present, the photo-adduct is not formed. Tyrosines are also quenchers for the flavin excited states, although not as efficient as Trp residues, as demonstrated with their substitution with the inert phenylalanine. For one of these variants, C71S/Y116F, we found that the quantum yield of formation for singlet oxygen is 0.44 in aqueous aerobic solution, vs 0.17 for C71S. Based on our study with Mr4511 and on literature data for other LOV domains we suggest that Trp and Tyr residues too close to the flavin chromophore (at distances less than 0.9 nm) reduce the yield of photoproduct formation and that introduction of inert Phe residues in key positions can help in developing efficient, LOV-based photosensitisers.

• A light life together: photosensing in the plant microbiota

Losi A.; Gärtner W.; Photochem Photobiol Sci, 20: 451–473 (2021) https://doi.org/10.1007/s43630-021-00029-7

Bacteria and fungi of the plant microbiota can be phytopathogens, parasites or symbionts that establish mutually advantageous relationships with plants. They are often rich in photoreceptors for UVA–Visible light, and in many cases, they exhibit light regulation of growth patterns, infectivity or virulence, reproductive traits, and production of pigments and of metabolites. In addition to the light-driven effects, often demonstrated via the generation of photoreceptor gene knock-outs, microbial photoreceptors can exert effects also in the dark. Interestingly, some fungi switch their attitude towards plants in dependence of illumination or dark conditions in as much as they may be symbiotic or pathogenic. This review summarizes the current knowledge about the roles of light and photoreceptors in plant-associated bacteria and fungi aiming at the identification of common traits and general working ideas. Still, reports on light-driven infection of plants are often restricted to the description of macroscopically observable phenomena, whereas detailed information on the molecular level, e.g., protein–protein interaction during signal transduction or induction mechanisms of infectivity/virulence initiation remains sparse. As it becomes apparent from still only few molecular studies, photoreceptors, often from the red- and the blue light sensitive groups interact and mutually modulate their individual effects. The topic is of great relevance, even in economic terms, referring to plant-pathogen or plant-symbionts interactions, considering the increasing usage of artificial illumination in greenhouses, the possible light-regulation of the synthesis of plant-growth stimulating substances or herbicides by certain symbionts, and the biocontrol of pests by selected fungi and bacteria in a sustainable agriculture.

• Reversible processes in collagen dehydration: A molecular dynamics study

Leo L.; Bridelli M.G.; Polverini E.; Archives of Biochemistry and Biophysics, 714: 109079 (2021) https://doi.org/10.1016/j.abb.2021.109079.

Collagen dehydration is an unavoidable damaging process that causes the lack of fibers’ physical properties and it is usually irreversible. However, the identification of low hydration conditions that permit a recovering of initial collagen features after a rehydration treatment is particularly of interest.
Monitoring structural changes by means of MD simulations, we investigated the hydration-dehydrationrehydration cycle of two microfibril models built on different fragments of the sequence of rat tail collagen type I. The microfibrils have different hydropathic features, to investigate the influence of amino acid composition on the whole process. We showed that with low hydration at a level corresponding to the first shell, microfibril gains in compactness and tubularity. Crucially, some water molecules remain trapped inside the fibrils, allowing, by rehydrating, a recovery of the initial collagen structural features. Water rearranges in cluster around the protein, and its first layer is more anchored to the surface. However, these changes in distribution and mobility in low hydration conditions get back with rehydration.

• Blue-Light Receptors for Optogenetics

Losi A.; Gardner K.H; Möglich A.; Chemical Reviews, 118(21): 10659–10709 (2018) https://doi.org/10.1021/acs.chemrev.8b00163.

Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

• Subdiffraction localization of a fluorescent photosensitizer in bacterial cells

Delcanale P.; Pennacchietti F.; Maestrini G.; Rodríguez-Amigo B.; Bianchini P.; Diaspro A.; Iagatti A.; Patrizi B.; Foggi P.; Agut M.; Nonell S.; Abbruzzetti S.; Viappiani C.; Sci Rep, 5: 15564 (2014) https://doi.org/10.1038/srep15564.

Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall, and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure.
 

• Experimental basis for a new allosteric model for multisubunit proteins

Viappiani C.; Abbruzzetti S.; Ronda L.; Bettati S.; Henry ER.; Mozzarelli A.; PNAS, 111(35): 12758-12763 (2014) https://doi.org/10.1073/pnas.1413566111.

Although the theoretical model of Monod, Wyman, and Changeux (MWC) is one of the most influential and highly cited theoreticalmodels in bioscience, it fails to explain allosteric effects in hemoglobin, the paradigm of allostery, because their model considers only quaternary preequilibria. By using a new kind of laser photolysis experiment, measurements of ligand rebinding kinetics for transient hemoglobin conformations trapped by encapsulation in silica gels support the simplest possible extension of the MWC allosteric model to include tertiary in addition to quaternary conformational preequilibria. While the MWC model provides a qualitative explanation for allostery in many multisubunit proteins, quantitative analysis will most probably require the extension used here to explain our new results.