Light induces oxidative damage and protein stability in the fungal photoreceptor Vivid

"Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus Neurospora crassa is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability.

La luz en la biología como una herramienta de medición y como un estímulo medioambiental

"Tanto en los organismos como en la biología las interacciones de la luz con la materia tienen varias consecuencias. Para los organismos, una de las consecuencias ha sido el desarrollo de proteínas fotorreceptoras, con las cuales los organismos pueden adaptase mejor a las condiciones impuestas por la radiación solar. Por otro lado, la biología actual se beneficia de las interacciones luz-materia por medio de técnicas ópticas como la microscopía, la espectroscopia y los ensayos de fluorescencia entre muchos otros. En este trabajo se presentan dos técnicas ópticas para el estudio de procesos biológicos y se describe un nuevo efecto de la luz azul sobre el fotorreceptor VIVID (VVD). Primero, se presenta un método para potenciar la microscopía de campo claro por medio del procesamiento digital de imágenes, el cual permite visualizar objetos de fase con suficiente contraste tal que, por primera vez, fue posible adquirir la función de dispersión de punto para objetos de fase (FDPf) y se mostró que es posible aplicar algoritmos de deconvolución estándar para mejorar la calidad de las imágenes de células no teñidas. En segundo lugar, se construyó un sistema de pinzas ópticas para el estudio de moléculas individuales. El correcto funcionamiento de nuestro sistema de pinzas ópticas fue validado por medio de dos ensayos estándar: se midieron las propiedades elásticas de una cadena de ADN y se midió la fuerza máxima ejercida y el tamaño de paso del motor molecular cinesina. En tercer lugar, se trabajó con la flavoproteína fotorreceptora de luz azul VVD, la cual pertenece al hongo filamentoso Neurospora crassa. Se mostró que en condiciones in vitro VVD presenta agregación al ser oxidada por especies reactivas de oxígeno producidas por la misma proteína. Encontramos que la sensibilidad de VVD a la luz, el oxígeno y la temperatura regulan la cinética de agregación de la proteína. Con base en nuestras observaciones dilucidamos la ruta de agregación de VVD y proponemos que su agregación podría tener efectos en el hongo N. crassa.""For organisms as for biology the light-matter interactions have several consequences. For organisms, one of these consequences has been the development of proteins specialized in light detection which provide organisms with a better fit to sunlight cues. On the other hand, current biology exploits the lightmatter interactions through optical techniques such as microscopy, spectroscopy, and fluorescence assays among many others. Herein two optical techniques to study biological processes and a new effect of blue light over the photoreceptor protein VIVID (VVD) are presented. First, the power of bright field light microscopy was enhanced by digital image processing, which allows us to visualize subdiffraction phase objects with enough contrast to acquire, for the first time, the phase point-spread function (PSFP) of a bright field light microscope. As a proof of concept, we used the measured PSFP to apply conventional deconvolution to bright field images, increasing image contrast and the definition of boundaries in unstained cellular samples. Second, an optical tweezers apparatus for singlemolecule studies was built and tested by performing two standard single-molecule assays: stretching single dsDNA molecules, and measuring the step size and maximum sustainable load for the molecular motor kinesin. Third, the flavin-binding blue-light (BL) photoreceptor VIVID from the filamentous fungus Neurospora crassa was shown to present an in vitro aggregation mechanism triggered by a self-produced reactive oxygen species (ROS). We found that VIVID (VVD) is sensitive to light, oxygen and temperature and these factors modulate the aggregation of the protein. Based on our observations we elucidate an aggregation pathway for VVD and propose aggregation as a possible mechanism to regulate diverse processes in N. crassa.

Direct Imaging of Phase Objects Enables Conventional Deconvolution in Bright Field Light Microscopy

<div><p>In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive) objects if the corresponding phase (or absorptive) impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments) serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.</p></div

Demonstration of deconvolution in the BF microscopy images of unstained, living <i>E. coli</i> cells.

<p>(A–C) BF images of bacteria before (“BF”) and after (“D”) deconvolution, together with their respective intensity profiles along the yellow dashed lines. Length of double-arrow lines in (B): m. (D) Image of a standing bacterium along three orthogonal slices (marked by the yellow dashed lines). In BF, the cell extends well beyond the position of the supporting coverslip (white dotted line), whereas the same views after deconvolution display the cell with improved definition of boundaries. (E) Time-lapse frames of a bacterium undergoing cell division under continuous illumination. No threshold or transparency levels were applied to deconvolved images. Scale bars: m.</p

Experimental OTF and comparison with theory.

<p>The modulus of the OTF is displayed (left) together with the result predicted by Eq. (5) (right), showing good agreement.</p

Direct measurement of the BF-PSF.

<p>(A) False-color images of a 100-nm bead at various axial positions. Field of view is 8.1 m8.1 m. (B) 3D view of the measured PSF. Arbitrary transparency and threshold levels were applied for display purposes. Field of view is 8.1 m8.1 m3.5 m.</p

A minimal optical trapping and imaging microscopy system

"We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (similar to 25 nm in diameter) and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.

Direct and indirect measurements of the BF-LSF.

<p>(A) A straight segment of an individual MT is chosen, shown at various defocusing positions. The corresponding pixel count profiles were obtained for each image by averaging all the pixel count values along a given pixel column. Scale bar: m. (B) The directly-measured LSF obtained from MT-profiles (false-color), together with intensity profiles. (C) The indirectly measured pLSF obtained from the experimental PSF (false-color), together with intensity profiles. (D) The tLSF derived from the theoretical PSF, together with intensity profiles. Profiles highlighted in black were used to determine main spot sizes. The intensities of the pLSF and tLSF were multiplied by arbitrary factors for comparison with the LSF.</p

Light induces oxidative damage and protein stability in the fungal photoreceptor Vivid

<div><p>Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus <i>Neurospora crassa</i> is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability.</p></div

The aggregation of VVD is under kinetic control and regulated by photoadduct dynamics.

<p>(A) Aggregation kinetics of VVD initially prepared in a dark-state or lit-state. The kinetics records are well fit (solid lines) by a second-order reaction model that considers photoadduct decay as the limiting step for VVD aggregation. Both samples were diluted in standard buffer (10% glycerol, 50 mM HEPES, 150 mM NaCl, 20 mM imidazole, pH 8). The lit-state corresponds to the same conditions tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201028#pone.0201028.g001" target="_blank">Fig 1B</a>. (B) Aggregation halts during the illumination cycling period and resumes only after samples are returned to the dark. Samples were initially illuminated for 30 s and their aggregation kinetics was followed. 5-s BL pulses were applied every ~5 min, over 68 min (circles) or 202 min (triangles). Aggregation kinetics of a sample subjected to only the initial BL pulse is included for reference (squares). Data: mean, error bars: SD; <i>n</i> = 3. (C-D), VVD lit-state is resistant to denaturant conditions whereas dark-state is not. Proteins initially prepared in a dark-state (C) or in a lit-state (D) were challenged with 0.01% SDS (black records) or left in standard buffer without SDS (gray records), and their absorption spectra were immediately acquired.</p