Biology subjects

Kucher, S.

Publications and source records attributed to Kucher, S..

5 recordsLinked to original sources

bioRxiv2026

Functional dynamics of the eukaryotic RNA exosome upon complex formation

Rather than being static, proteins adopt multiple conformations and undergo dynamics on a wide range of timescales. Such conformational changes can be routinely probed by solution state NMR for protein sizes below 40 kDa and can be extended to large protein complexes with selective methyl-labeling strategies. Here, we expand the limit of methyl-NMR dynamics experiments to asymmetric protein complexes bigger than 300 kDa by investigating the inert core of the eukaryotic RNA exosome, Exo9. We demonstrate that isolated subunits are highly dynamic on the ms-s dynamics, which facilitates assembly. While ring subunits lose dynamics upon complex-formation, cap subunits and functional loops retain a large degree of flexibility as demonstrated by relaxation dispersion and chemical exchange saturation transfer experiments in the fully asymmetric Exo9 complex. Employing EPR spectroscopy we additionally revealed changes in the conformational ensemble and nanosecond dynamics of a spin-labeled functional loop region in the presence of the catalytic subunit Rrp44 and in response to substrate RNA binding. We thus demonstrate that an integrated NMR-EPR approach facilitates the characterization of functional dynamics that occur on a wide range of timescales in very large protein complexes.

biophysics↗

bioRxiv2024

A combined approach to extract rotational dynamics of globular proteins undergoing liquid-liquid phase separation

The formation of protein condensates (droplets) via liquid-liquid phase separation (LLPS) is a commonly observed phenomenon in vitro. Changing the environmental properties with cosolutes, molecular crowders, protein partners, temperature, pressure, etc. was shown to favour or disfavour the formation of protein droplets by fine-tuning the water-water, water-protein and protein-protein interactions. Therefore, these environmental properties and their spatiotemporal fine-tuning are likely to be important also in a cellular context at the existing protein expression levels. One of the key physicochemical properties of biomolecules impacted by molecular crowding is diffusion, which determines the viscoelastic behaviour of the condensates. Here we investigate the change in the rotational diffusion of {gamma}D-crystallin, undergoing LLPS in vitro in aqueous solutions in absence and presence of cosolutes. We studied its rotational dynamics using molecular dynamics simulations (MD), electron paramagnetic resonance (EPR) spectroscopy and fluorescence spectroscopy. MD simulations performed under dilute and crowded conditions show that the rotational diffusion of crystallin in water is retarded by one to two orders of magnitude in the condensed phase. To obtain the rotational dynamics in the dilute phase we used fluorescence anisotropy and to extract the retardation factor in the condensed phase we used spin-labeled {gamma}D-crystallin proteins as EPR viscosity nanoprobes. Aided by a viscosity nanoruler calibrated with solutions at increasing sucrose concentrations, we validate the rotational diffusion retardation predicted by MD simulations. This study underlines the predictive power of MD simulations and showcases the use of a sensitive EPR nanoprobe to extract the viscosity of biomolecular condensates.

biophysics↗

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↗

bioRxiv2022

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex Photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen-evolving activity of approx. 6,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen-evolving activity of approx. 3,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1 and manganese content of 2.7 ({+/-} 0.2) per reaction center compared to 3.7 ({+/-} 0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role in the repair of the complex.

plant biology↗

bioRxiv2018

The extracellular gate shapes the energy profile of an ABC exporter

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 [A] crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporters conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

biochemistry↗