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Register a new userMonitoring the conformational dynamics of a single potassium transporter by ALEX-FRET
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Conformational changes of single proteins are monitored in real time by Forster-type resonance energy transfer, FRET. Two different fluorophores have to be attached to those protein domains, which move during function. The distance between the fluorophores is measured by relative fluorescence intensity changes of FRET donor and acceptor fluorophore, or by fluorescence lifetime changes of the FRET donor. The fluorescence spectrum of a single FRET donor fluorophore is influenced by local protein environment dynamics causing apparent fluorescence intensity changes on the FRET donor and acceptor detector channels. To discriminate between those spectral fluctuations and distance-dependent FRET, alternating pulsed excitation schemes (ALEX) have recently been introduced which simultaneously probe the existence of a FRET acceptor fluorophore. Here we employ single-molecule FRET measurements to a membrane protein. The membrane-embedded KdpFABC complex transports potassium ions across a lipid bilayer using ATP hydrolysis. Our study aims at the observation of conformational fluctuations within a single P-type ATPase functionally reconstituted into liposomes by single-molecule FRET and analysis by Hidden-Markov-Models.
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FoF1-ATP synthase is the enzyme that provides the chemical energy currency adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound Fo part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the gamma and epsilon subunits of the F1 sector resulting in 120 degree steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F1 motor, another one to the Fo motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of Fo and F1.
The totally asymmetric simple exclusion process (TASEP), which describes the stochastic dynamics of interacting particles on a lattice, has been actively studied over the past several decades and applied to model important biological transport processes. Here we present a software package, called EGGTART (Extensive GUI gives TASEP-realization in real time), which quantifies and visualizes the dynamics associated with a generalized version of the TASEP with an extended particle size and heterogeneous jump rates. This computational tool is based on analytic formulas obtained from deriving and solving the hydrodynamic limit of the process. It allows an immediate quantification of the particle density, flux, and phase diagram, as a function of a few key parameters associated with the system, which would be difficult to achieve via conventional stochastic simulations. Our software should therefore be of interest to biophysicists studying general transport processes, and can in particular be used in the context of gene expression to model and quantify mRNA translation of different coding sequences.
FoF1-ATP synthase is the enzyme that provides the chemical energy currency adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. We monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using two fluorophores specifically attached to the enzyme. To identify the stepsize of rotary movements by the motors of ATP synthase we simulated the confocal single-molecule FRET data of freely diffusing enzymes and developed a step finder algorithm based on Hidden Markov Models (HMM). The HMM is able to find the proximity factors, P, for a three-level system and for a five-level system, and to unravel the dwell times of the simulated rotary movements. To identify the number of hidden states in the system, a likelihood parameter is calculated for the series of one-state to eight-state HMMs applied to each set of simulated data. Thereby, the basic prerequisites for the experimental single-molecule FRET data are defined that allow for discrimination between a 120 degree stepping mode or a 36 degree substep rotation mode for the proton-driven Fo motor of ATP synthase.
An improved analysis for single particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the AMO instrument at the Linac Coherent Light Source (LCLS) as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from the half of the detector and small fraction of single hits. General SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus structure determination step. The presented processing pipeline allowed us to determine the three-dimensional structure of the bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9 nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits.
Confocal time resolved single-molecule spectroscopy using pulsed laser excitation and synchronized multi channel time correlated single photon counting (TCSPC) provides detailed information about the conformational changes of a biological motor in real time. We studied the formation of adenosine triphosphate, ATP, from ADP and phosphate by FoF1-ATP synthase. The reaction is performed by a stepwise internal rotation of subunits of the lipid membrane-embedded enzyme. Using fluorescence resonance energy transfer, FRET, we detected rotation of this biological motor by sequential changes of intramolecular distances within a single FoF1-ATP synthase. Prolonged observation times of single enzymes were achieved by functional immobilization to the glass surface. The stepwise rotary subunit movements were identified by Hidden Markov Models (HMM) which were trained with single-molecule FRET trajectories. To improve the accuracy of the HMM analysis we included the single-molecule fluorescence lifetime of the FRET donor and used alternating laser excitation to co-localize the FRET acceptor independently within a photon burst. The HMM analysis yielded the orientations and dwell times of rotary subunits during stepwise rotation. In addition, the action mode of bactericidal drugs, i.e. inhibitors of FoF1-ATP synthase like aurovertin, could be investigated by the time resolved single-molecule FRET approach.
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