Biology subjects

Skalidis, I.

Publications and source records attributed to Skalidis, I..

4 recordsLinked to original sources

bioRxiv2025

Direct evidence of acid-driven protein desolvation

Water and its ability to modulate the protonation states of biomolecules govern the physical chemistry of life, dictating their metabolic functions(1). However, how amino acid protonation alters protein hydration and solubility(2, 3) is an open question since Kuntz and Kauzmann proposed pH-driven protein desolvation in 1974(4). Here, in a series of high-resolution cryo-electron microscopy structures of a protein complex at different pH values (from pH 9.0 to 3.5) we examined thousands of observable hydration sites. Cryo-EM data, in agreement with constant-pH molecular dynamics simulations, show that nearly half of protein-bound waters exchanged with the bulk solvent upon acidification, with [~]100 waters lost per pH unit per molecule. The loss of waters was most significant around the side chains of glutamate and aspartate residues while specific polar residues, mostly asparagine, anchored persistent waters. A positionally conserved hydration layer was observed across all pH conditions, accounting for 40% of resolved waters. Those waters displayed denser packing than less persistent waters, forming a pH-independent solvation shell. Acid-induced water exchange also displaced bound iron, providing a mechanistic link between solvation and metal release. Our findings demonstrate the core principles of acid-driven protein desolvation, resolving a 50-year-old biochemical hypothesis(4).

biochemistry↗

bioRxiv2025

Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus

Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 [A] structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.

molecular biology↗

bioRxiv2022

AI-guided cryo-EM probes a thermophilic cell-free system with succinyl-coA manufacturing capability

Cell-free systems display tremendous potential for biotechnological applications, complementing in vitro reconstituted enzymatic processes and traditional expression systems. However, they often represent "black boxes" without much insight into their components. Here, we characterize a thermophilic cell-free system that produces succinyl-CoA and discern its intrinsic, non-stochastic organization. By employing biochemical, biophysical, and bioinformatic methods we resolve its molecular composition, 3D architecture and molecular function at atomic resolution. We further report the high-resolution cryo-EM structure of the reactions main component, the oxoglutarate dehydrogenase complex core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-communication between subunits, and the presence of a flexible subunit, the E3BPo connecting E2o and E3. This multi-scale analysis of a cell-free system provides a blueprint for structure-function studies of complex mixtures of biotechnological value.

biochemistry↗

bioRxiv2019

2.7 A cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV \"screening microscope\"

Here we present the structure of mouse H-chain apoferritin at 2.7 [A] (FSC=0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device. Data were collected using a compact, two-lens illumination system with a constant power objective lens, without the use of energy filters or aberration correctors. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (x-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving resolution close to the Rayleigh limit. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging, widely thought of as a "screening cryo-microscope", can be used to determine structures approaching atomic resolution. HighlightsO_LIThe 2.7 [A] structure of mouse apoferritin was solved using a 200 keV screening cryo-microscope C_LIO_LIThe apoferritin reconstruction was resolved without an energy filter, aberration correctors, or constant-power condenser lenses C_LIO_LIComparison to available crystallographic and cryo-EM structures from high-end cryo-microscopes demonstrates consistency in resolved water molecules, metals and side chain orientations C_LIO_LIAlthough radiation damage is more prominent at 200 keV compared to 300 keV, this type of instrumentation is more accessible to research laboratories due to its compactness and simplicity C_LI

biophysics↗