20 research outputs found
Photochromism of flavin-based photoreceptors: new perspectives for super-resolution microscopy and optogenetics
Flavin-binding blue-light (BL) sensor proteins (Fl-Blues) of bacterial origin have been investigated in this work with diverse biophysical methods, with the aim to characterize their photophysical and photochemical properties. Investigations have been carried out both in vitro and in vivo, to explore their potential in advance cellular applications and establish novel methods of analysis. Besides the lab work, extensive in silico analysis have been performed, in the attempt to optimize searching in public databanks, identify novel Fl-Blues and gain hints on their evolution patterns.
Main object of lab work has been the protein YtvA from Bacillus subtilis, a BL-photoreceptor involved in environmental stress responses. Its photosensing unit is a LOV (Light, Oxygen and Voltage) domain, that binds oxidized flavin mononucleotide (FMN) as a photoactive chromophore, brightly fluorescent in the dark-adapted state. BL illumination triggers a photocycle with formation of a FMN-cysteine covalent adduct (light adapted or lit state), with complete loss of fluorescence and concomitant activation of a biological response (signaling). The adduct thermally recovers to the dark-state with breakage of the covalent bond, on a time scale of hours at room temperature. This protein is thus naturally bi-stable, both for its optical and biological properties.
Using steady-state and time resolved optical spectroscopies, we could show here that application of violet or UVA light to the adduct is able to induce a photoequilibrium, with partial recovery of the dark-adapted state and of FMN fluorescence. In other words, YtvA was demostrated to be a photochromic system. It was possible to calculate the quantum yield of the photoinduced light-to-dark reaction as ca. 0.05, that is about 1/10 the efficiency of the forward dark-to-light photochemical step. This newly discovered photochromism of YtvA, allowed us to show its potential in super-resolution microscopy, in particular using Fluorescence Photo-activation Localization Microscopy (FPALM) to visualize YtvA fluorescence inside transformed Escherichia coli cells with nanometer precision. In order to try to optimize and tune the photochromic, photochemical and spectral properties of YtvA, a set of mutagenized variants has been designed and investigated. In particular we were able to identify amino acids that act as major spectral tuners in the UVA region, and other residues able to strongly affects the dynamics of the photocycle.
Another notable property of YtvA has emerged by performing epifluorescence microscopy experiments on living cells expressing the proteins. During these studies, still at a preliminary stage, it became clear that the level of hydration is able to affect the thermal recovery rate of the photocycle, a property that is relevant to understand the effects of water on this kind of systems and for applications in vivo.
Genome digging has revealed, during the last decade, that LOV domains are widespread among the three life domains and are emerging as BL sensing systems not only in plants, but also in prokaryotes. On full-length proteins LOV modules they are usually linked to diverse effector domains that determine the biological functionality of the protein itself. A second and different type of BL-sensor, also widespread among prokaryotes, is the so-called BLUF (Blue Light sensing Using Flavins), that upon light-activation undergoes a rapidly reversible hydrogen-bond (HB) switch. In this work we performed an extensive database search in order to have a comprehensive scenario of LOV and BLUF proteins in the prokaryotic world, inspect phylogenetic pathways and discover possible novel functionalities for effector domains, a feature connected with optogenetics. From distance-trees obtained by using neighbouring-methods, it was observed that most LOV and BLUF domains from organisms belonging to the same phylum are neighbour, but that in many cases clustering occurs according to effector functions associated to the photosensing domains. Metagenomics and bio-informatic analysis have only recently been initated, but signatures are beginning to emerge that allow definition of a bona fide LOV or BLUF domain, aiming at better selection criteria for novel BL sensors based on sequence logos. Taking advantage of these new criteria it was built, for the first time, the phylogenetic tree for archaeal LOV domains that have reached a statistically significant number, but have not at all been investigated so far
The Evolution and Functional Role of Flavin-based Prokaryotic Photoreceptors
Flavin-based photoreceptor proteins of the LOV (light, oxygen
and voltage) superfamily are ubiquitous and appear to be
essential blue-light sensing systems not only in plants, algae
and fungi, but also in prokaryotes, where they are represented
in more than 10% of known species. Despite their broad occurrence,
only in few cases LOV proteins have been correlated
with important phenomena such as bacterial infectivity, selective
growth patterns or/and stress responses; nevertheless these
few known roles are helping us understand the multiple ways
by which prokaryotes can exploit these soluble blue-light photoreceptors.
Given the large number of sequences now deposited
in databases, it becomes meaningful to define a signature
for bona fide LOV domains, a procedure that facilitates identification
of proteins with new properties and phylogenetic analysis.
The latter clearly evidences that a class of LOV proteins
from alpha-proteobacteria is the closest prokaryotic relative of
eukaryotic LOV domains, whereas cyanobacterial sequences
cluster with the archaeal and the other bacterial LOV
domains. Distance trees built for LOV domains suggest complex
evolutionary patterns, possibly involving multiple horizontal
gene transfer events. Based on available data, the in vivo
relevance and evolution of prokaryotic LOV is discussed
From Plant Infectivity to Growth Patterns: The Role of Blue-Light Sensing in the Prokaryotic World
Flavin-based photoreceptor proteins of the LOV (Light, Oxygen, and Voltage) and BLUF (Blue Light sensing Using Flavins) superfamilies are ubiquitous among the three life domains and are essential blue-light sensing systems, not only in plants and algae, but also in prokaryotes. Here we review their biological roles in the prokaryotic world and their evolution pathways. An unexpected large number of bacterial species possess flavin-based photosensors, amongst which are important human and plant pathogens. Still, few cases are reported where the activity of blue-light sensors could be correlated to infectivity and/or has been shown to be involved in the activation of specific genes, resulting in selective growth patterns. Metagenomics and bio-informatic analysis have only recently been initiated, but signatures are beginning to emerge that allow definition of a bona fide LOV or BLUF domain, aiming at better selection criteria for novel blue-light sensors. We also present here, for the first time, the phylogenetic tree for archaeal LOV domains that have reached a statistically significant number but have not at all been investigated thus far
The amino acids surrounding the flavin 7a-methyl group determine the UVA spectral features of a LOV protein
Flavin-binding light, oxygen, and voltage (LOV)
domains are UVA/blue-light-sensing protein units that
form a reversible flavin mononucleotide-cysteine adduct
upon light induction. In their dark-adapted state, LOV
domains exhibit the typical spectral features of fully
oxidized riboflavin derivatives. A survey on the absorption
spectra of various LOV domains revealed that the
UVA spectral range is the most variable region (whereas
the absorption band at 450 nm is virtually unchanged),
showing essentially two distinct patterns found in plant
phototropin LOV1 and LOV2 domains, respectively. In this
work, we have identified a residue directly interacting
with the isoalloxazine methyl group at C(7a) as the major
UVA spectral tuner. In YtvA from Bacillus subtilis, this
amino acid is threonine 30, and its mutation into apolar
residues converts the LOV2-like spectrum of native YtvA
into a LOV1-like pattern. Mutation T30A also accelerates
the photocycle ca. 4-fold. Together with control mutations
at different positions, our results experimentally confirm
the previously calculated direction of the transition dipole
moment for the UVA ππ* state and identify the mechanisms
underlying spectral tuning in the LOV domains
A Photochromic Bacterial Photoreceptor with Potential for Super-Resolution Microscopy
We introduce a novel fluorescent reporter with potential for super-resolution microscopy, based on the bacterial photoreceptor YtvA. YtvA (from Bacillus subtilis) comprises a photosensitive flavin based LOV domain, efficiently photoswitchable between fluorescent and non-fluorescent states.
We demonstrate Fluorescence PhotoActivation Localization Microscopy (FPALM) studies of live Escherichia coli cells, expressing YtvA molecules
Apparent time constants for the YtvAL to YtvAD relaxation at 25°C.
<p>Apparent time constants for the YtvAL to YtvAD relaxation at 25°C.</p
Three dimensional arrangement of mutated amino acids in YtvA.
<p>Left. Closeup of the YtvA chromophore (in green) with the amino acids considered in this study represented in capped sticks (red oxygen, blue nitrogen). Right. Solvent accessible surface visualization of the protein with the chromophore in green and the amino acids considered in this study represented in capped sticks.</p
Dark recovery kinetics of dried YtvA.
<p><b>A.</b> Absorption spectrum for YtvA molecules air-dried on a quartz plate before (black) and after (red) photoconversion with LED465. The green curve reports the absorption spectrum at t = 24660 s at 25°C. After 24 hours the YtvAD spectrum is fully recovered (not shown). <b>B.</b> Dark recovery kinetics of YtvAL to YtvAD followed through the absorbance at 475 nm. The red solid line is the best fit with a double stretched exponential relaxation, with τ = 600±400 s and β = 0.6±0.2 (16%) and τ = (3.7±0.3)×10<sup>4 </sup>s and β = 1.0±0.2 (84%).</p