54 research outputs found
Insights and approaches
Fluorescence spectroscopy has become an established tool at the interface of
biology, chemistry and physics because of its exquisite sensitivity and recent
technical advancements. However, rhodopsin proteins present the fluorescence
spectroscopist with a unique set of challenges and opportunities due to the
presence of the light-sensitive retinal chromophore. This review briefly
summarizes some approaches that have successfully met these challenges and the
novel insights they have yielded about rhodopsin structure and function. We
start with a brief overview of fluorescence fundamentals and experimental
methodologies, followed by more specific discussions of technical challenges
rhodopsin proteins present to fluorescence studies. Finally, we end by
discussing some of the unique insights that have been gained specifically
about visual rhodopsin and its interactions with affiliate proteins through
the use of fluorescence spectroscopy. This article is part of a Special Issue
entitled: Retinal Proteins - You can teach an old dog new tricks
Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins
We studied the activity of the fluorescently labeled membrane transporter
MalGFK2, which transports maltose at the expense of ATP hydrolysis. We used a
commercially available malachite green assay (SensoLyte MG phosphate assay
kit; Anaspec) to quantify the liberated phosphate upon ATP hydrolysis.
However, strong variations in phosphate concentration were measured when using
the supplier’s handling protocol. We optimized the protocol, taking into
account the effects mediated by glycerol, SDS, and fluorescent label on the
sample. As a result we obtained highly reproducible phosphate concentration
values under conditions optimal for solubilized membrane proteins
Activation and molecular recognition of the GPCR rhodopsin – Insights from time-resolved fluorescence depolarisation and single molecule experiments
The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is
a key element in membrane receptor activation, molecular recognition by
signalling molecules, and receptor deactivation. Understanding of the coupling
between conformational changes in the intramembrane domain and the membrane-
exposed surface of the photoreceptor rhodopsin is crucial for the elucidation
of the molecular mechanism in GPCR activation. As little is known about
protein dynamics, particularly the conformational dynamics of the cytoplasmic
surface elements on the nanoseconds timescale, we utilised time-resolved
fluorescence anisotropy experiments and site-directed fluorescence labelling
to provide information on both, conformational space and motion. We summarise
our recent advances in understanding rhodopsin dynamics and function using
time-resolved fluorescence depolarisation and single molecule fluorescence
experiments, with particular focus on the amphipathic helix 8, lying parallel
to the cytoplasmic membrane surface and connecting transmembrane helix 7 with
the long C-terminal tail
Diffusion Analysis of NAnoscopic Ensembles: A Tracking-Free Diffusivity Analysis for Nanoscopic Ensembles in Biological Samples and Nanotechnology
The rapid development of microscopic techniques over the past decades enables the establishment of single molecule fluorescence imaging as a powerful tool in biological and biomedical sciences. Single molecule fluorescence imaging allows to study the chemical, physicochemical, and biological properties of target molecules or particles by tracking their molecular position in the biological environment and determining their dynamic behavior. However, the precise determination of particle distribution and diffusivities is often challenging due to high molecule/particle densities, fast diffusion, and photobleaching/blinking of the fluorophore. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of NAnoscopic Ensembles (DANAE), that solves all these obstacles is introduced. DANAE requires no approximations or any a priori input regarding unknown system-inherent parameters, such as background distributions; a requirement that is vitally important when studying the behavior of molecules/particles in living cells. The superiority of DANAE with various data from simulations is demonstrated. As experimental applications of DANAE, membrane receptor diffusion in its natural membrane environment, and cargo mobility/distribution within nanostructured lipid nanoparticles are presented. Finally, the method is extended to two-color channel fluorescence microscopy
Long-Distance Protonation-Conformation Coupling in Phytochrome Species
Phytochromes are biological red/far-red light sensors found in many organisms. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY, PGP) and the C-terminal transmitter (output) module. We recently showed a direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains of the prototypical phytochrome Cph1 PGP. These results suggested that the transient phycocyanobilin (PCB) chromophore deprotonation is closely associated with a higher protein mobility both in proximal and distal protein sites, implying a causal relationship that might be important for the global large-scale protein rearrangements. Here, we investigate the prototypical biliverdin (BV)-binding phytochrome Agp1. The structural changes at various positions in Agp1 PGP were investigated as a function of pH using picosecond time-resolved fluorescence anisotropy and site-directed fluorescence labeling of cysteine variants of Agp1 PGP. We show that the direct correlation of chromophore deprotonation with pH-dependent conformational changes does not occur in Agp1. Together with the absence of long-range effects between the PHY domain and chromophore pKa, in contrast to the findings in Cph1, our results imply phytochrome species-specific correlations between transient chromophore deprotonation and intramolecular signal transduction
Picosecond Multidimensional Fluorescence Spectroscopy: A Tool to Measure Real-time Protein Dynamics During Function â€
ABSTRACT Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico-to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is lightactivated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPCmodule. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket
A simulation-guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase
Fluorescence correlation spectroscopy (FCS) is a single molecule based
technique to temporally resolve rate-dependent processes by correlating the
fluorescence fluctuations of individual molecules traversing through a
confocal volume. In addition, chemical processes like protonation or
intersystem crossing can be monitored in the sub-microsecond range. FCS
thereby provides an excellent tool for investigations of protonation dynamics
in proton pumps like cytochrome c oxidase (CcO). To achieve this, the pH-
dependent fluorescent dye fluorescein was attached as a protonation probe to
the CcO surface via site-specific labeling of single reactive cysteines that
are located close to the entry point of a proton input channel (K-pathway).
The analysis of protonation dynamics is complicated by overlapping triplet and
protonation rates of the fluorophore. A Monte Carlo simulation based algorithm
was developed to facilitate discrimination of these temporally overlapping
processes thus allowing for improved protonation reaction rate determination.
Using this simulation-guided approach we determined precise local proton
association and dissociation rates and provide information about protein
surface effects, such as proton collecting antennae, on the transport
properties of proton transfer channels
Osmolytes Modulate Photoactivation of Phytochrome: Probing Protein Hydration
Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials
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