Biology subjects

Seidel, C. A. M.

Publications and source records attributed to Seidel, C. A. M..

6 recordsLinked to original sources

bioRxiv2024

Multi-state kinetics of the syringe-like injection mechanism of Tc toxins

Tc toxins are virulence factors of many insects and human pathogenic bacteria. They attach as soluble prepores to receptors on host cells and following acidification in the late endosome, perforate the cell membrane like a syringe to translocate toxic enzymes into the host cell through their pore-forming channel. Although this complex transformation has been structurally well studied, the functional aspects of this large-scale rearrangement, such as the reaction pathway with possible intermediate states and the resulting temporal evolution have remained elusive. Here, we used an integrated biophysical approach to monitor the prepore-to-pore transition and found that it takes [~]28 h when induced by high pH in the absence of other factors. In the presence of liposomes, an increasingly high pH or receptors, such as heparin or Vsg, the probability to transform prepores to pores increases by a factor of up to 4. This effect can also be mimicked by biotinylation or site-directed mutagenesis of the shell, demonstrating that shell destabilization is a crucial step in prepore-to-pore transition. We show that shell opening is a heterogeneous process with transition times ranging from 60 ms to 1.6 s and resolve three sequential intermediate states: an initial transient intermediate during shell destabilization, a first stable intermediate where the receptor-binding domains on the shell rearrange and a second stable intermediate with an open shell. In contrast, the ejection of the pore-forming channel from the open shell is highly cooperative with a transition time of < 60 ms. This detailed knowledge of the Tc toxin mechanism of action, even in the absence of receptors, is important for the future application of Tc toxins as biomedical devices or biopesticides.

biochemistry↗

bioRxiv2023

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

biophysics↗

bioRxiv2022

A Minimal Model of CD95 Signal Initiation Revealed by Advanced Super-resolution and Multiparametric Fluorescence Microscopy

Unraveling the spatiotemporal organization and dynamical interactions of receptors in the plasma membrane remains a key challenge for our mechanistic understanding of cell signal initiation. A paradigm of such process is the oligomerization of TNF receptor CD95 during apoptosis signaling, where molecular configurations are yet to be defined. Here, we scrutinize proposed oligomerization models in live cells, establishing a molecular sensitive imaging toolkit including time-resolved FRET spectroscopy, quantitative STED microscopy, confocal Photobleaching Step Analysis and FCS. CD95 interactions were probed over molecular concentrations of few to [~] 1000 molecules/{micro}m2, over ns to hours, and molecular to cellular scales. We further established high-fidelity monomer and dimer controls for quantitative benchmarking. Efficient apoptosis was already observed when [~] 8 to 17% monomeric CD95 oligomerize to dimers/trimers after ligand binding. Our multiscale study highlights the importance of molecular concentrations, of the native environment, and reveals a minimal oligomerization model of CD95 signal initiation.

biophysics↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2022

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation, especially at low endogenous concentrations found in cells, are thought to follow the tenets of classical nucleation theory describing a sharp transition between a dense phase and a dilute phase characterized by dispersed monomers. Here, we used in vitro biophysical studies to study subsaturated solutions of phase separating RNA binding proteins with intrinsically disordered prion like domains (PLDs) and RNA binding domains (RBDs). Surprisingly, we find that subsaturated solutions are characterized by heterogeneous distributions of clusters comprising tens to hundreds of molecules. These clusters also include low abundance mesoscale species that are several hundreds of nanometers in diameter. Our results show that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. Interestingly, however, cluster formation and phase separation can be decoupled from one another using solutes that impact the solubilities of phase separating proteins. They can also be decoupled by specific types of mutations. Overall, our findings implicate the presence of distinct, sequence-specific energy scales that contribute to the overall phase behaviors of RNA binding proteins. We discuss our findings in the context of theories of associative polymers. Significance StatementMembraneless biomolecular condensates are molecular communities with distinct compositional preferences and functions. Considerable attention has focused on phase separation as the process that gives rise to condensates. Here, we show that subsaturated solutions of RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions. The formation of clusters in subsaturated solutions and condensates in supersaturated solution are coupled through sequence-specific interactions. Given the low endogenous concentrations of phase separating proteins, our findings suggest that clusters in subsaturated conditions might be of functional relevance in cells.

biophysics↗

bioRxiv2021

Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

biophysics↗