Sonja Schmid

Functional Characterization of the Major and Minor Phosphorylation Sites of the P Protein of Borna Disease Virus

ABSTRACT The phosphoprotein P of Borna disease virus (BDV) is an essential cofactor of the viral RNA-dependent RNA polymerase. It is preferentially phosphorylated at serine residues 26 and 28 by protein kinase C ε (PKCε) and, to a lesser extent, at serine residues 70 and 86 by casein kinase II (CKII). To determine whether P phosphorylation is required for viral polymerase activity, we generated P mutants lacking either the PKCε or the CKII phosphate acceptor sites by replacing the corresponding serine residues with alanine (A). Alternatively, these sites were replaced by aspartic acid (D) to mimic phosphorylation. Functional characterization of the various mutants in the BDV minireplicon assay revealed that D substitutions at the CKII sites inhibited the polymerase-supporting activity of P, while A substitutions maintained wild-type activity. Likewise, D substitutions at the PKC sites did not impair the cofactor function of BDV-P, whereas A substitutions at these sites led to increased activity. Interestingly, recombinant viruses could be rescued only when P mutants with modified PKCε sites were used but not when both CKII sites were altered. PKCε mutant viruses showed a reduced capacity to spread in cell culture, while viral RNA and protein expression levels in persistently infected cells were almost normal. Further mutational analyses revealed that substitutions at individual CKII sites were, with the exception of a substitution of A for S86, detrimental for viral rescue. These data demonstrate that, in contrast to other viral P proteins, the cofactor activity of BDV-P is negatively regulated by phosphorylation.

Mutation of the Protein Kinase C Site in Borna Disease Virus Phosphoprotein Abrogates Viral Interference with Neuronal Signaling and Restores Normal Synaptic Activity

Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV) represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS) and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC)–dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P) may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.

A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously.

Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other. Multicolor smFRET experiments enable the direct observation of such correlated dynamics and interactions. Here we demonstrate the power and limitations of multicolor smFRET experiments including the description of a multicolor smFRET setup and data analysis. A general analytical procedure for multicolor smFRET data is presented and applied to the multicomponent heat shock protein 90 system. This allows us to identify microscopic states in transient complexes. Conformational dynamics and nucleotide binding are simultaneously detected, which is impossible using two-color smFRET. Additionally, their correlation is quantified using 3D ensemble hidden Markov analysis, in and out of equilibrium. This method is perfectly suited for protein systems that are much more sophisticated than previously studied DNA-based systems. By extending the application to biologically relevant systems, multicolor smFRET comes of age and provides a unique mechanistic insight into protein machines.

Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium

We present a simple and robust technique for extracting kinetic rate models and thermodynamic quantities from single-molecule time traces. Single-molecule analysis of complex kinetic sequences (SMACKS) is a maximum-likelihood approach that resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete data set while allowing for experimental variations between individual trajectories. In contrast to dwell-time analysis, which is the current standard method, SMACKS includes every experimental data point, not only dwell times. As a result, it works as well for long trajectories as for an equivalent set of short ones. In addition, the previous systematic overestimation of fast over slow rates is solved. We demonstrate the power of SMACKS on the kinetics of the multidomain protein Hsp90 measured by single-molecule Förster resonance energy transfer. Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90s ATPase function. SMACKS resolves accurate rate models even if states cause indistinguishable signals. Thereby, it pushes the boundaries of single-molecule kinetics beyond those of current methods.

Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.A multi-laboratory study finds that single-molecule FRET is a reproducible and reliable approach for determining accurate distances in dye-labeled DNA duplexes.

Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings-such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.

Effects of Inhibitors on Hsp90′s Conformational Dynamics, Cochaperone and Client Interactions

The molecular chaperone and heat-shock protein Hsp90 has become a central target in anti-cancer therapy. Nevertheless, the effect of Hsp90 inhibition is still not understood at the molecular level, preventing a truly rational drug design. Here we report on the effect of the most prominent drug candidates, namely, radicicol, geldanamycin, derivatives of purine, and novobiocin, on Hsp90s characteristic conformational dynamics and the binding of three interaction partners. Unexpectedly, the global opening and closing transitions are hardly affected by Hsp90 inhibitors. Moreover, we find no significant changes in the binding of the cochaperones Aha1 and p23 nor of the model substrate Δ131Δ. This holds true for competitive and allosteric inhibitors. Therefore, direct inhibition mechanisms affecting only one molecular interaction are unlikely. We suggest that the inhibitory action observed in vivo is caused by a combination of subtle effects, which can be used in the search for novel Hsp90 inhibition mechanisms.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Experiment-friendly kinetic analysis of single molecule data in and out of equilibrium

We present a simple and robust technique to extract kinetic rate models and thermodynamic quantities from single molecule time traces. SMACKS (Single Molecule Analysis of Complex Kinetic Sequences) is a maximum likelihood approach that works equally well for long trajectories as for a set of short ones. It resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete dataset, while allowing for experimental variations between individual trajectories. In particular, neither a priori models nor equilibrium have to be assumed. The power of SMACKS is demonstrated on the kinetics of the multi-domain protein Hsp90 measured by smFRET (single molecule Förster resonance energy transfer). Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90’s ATPase function. SMACKS pushes the boundaries of single molecule kinetics far beyond current methods.

Quantitative Protein Kinetics from sm-FRET Time Traces

Single molecule time traces (from fluorescence or force spectroscopy, nanopores, ion channels, etc.) provide unique information on the kinetic state sequence of the system under study.However, single molecule Foerster resonance energy transfer (smFRET) data is often used to uncover equilibrium distributions only, leaving the kinetic information unexploited. This is because of experimental limitations associated with smFRET, such as photo-bleaching, inter-fluorophore variation and the limited time window. Therefore, analysis tools beyond dwell-time histograms are required for smFRET time traces.Here we present a new maximum likelihood-based approach custom-tailored for experimental smFRET data. Our analysis tool handles multiple input traces, experimental inter-fluorophore variation, truly hidden states, and performs unbiased model selection. We demonstrate the robustness of rate determination and model selection on the basis of experimental and simulated data.Finally, we solve the kinetic state model for the complex conformational dynamics of the heat shock protein Hsp90 with unprecedented accuracy. This reveals novel insights into the effects of drug candidates, nucleotides and co-chaperones on Hsp90s kinetics.