Biology subjects

Argudo, P. G.

Publications and source records attributed to Argudo, P. G..

3 recordsLinked to original sources

bioRxiv2026

The structured hairpin region of the bacterial ESCRT-III protein IM30 orchestrates stress-induced condensate formation

Biomolecular condensates are well characterized in eukaryotes, but their role in bacteria remains largely elusive. In the cyanobacteriumn Synechocystis sp. PCC 6803, the protein IM30, a member of the ESCRT-III superfamily of membrane remodeling proteins, forms stress-induced puncta across diverse environmental challenges, indicating a general adaptive response. Live-cell imaging reveals that IM30 is uniformly distributed throughout the cytoplasm at low concentrations but assembles into puncta upon exceeding a critical saturation threshold, a hallmark of liquid-liquid phase separation. Crucially, stress triggers puncta formation even below this threshold, suggesting stress lowers the phase-separation barrier. Super-resolution microscopy confirms spherical, condensate-like morphologies, while FRAP demonstrates rapid fluorescence recovery, consistent with with a fluid interior and dynamic exchange between puncta and the cytosol. Cellular IM30 levels exceed the in vitro determined critical concentration, placing the protein in a supersaturated state primed for condensation. Domain mapping identifies the structured 1-3 helical hairpin as the minimal phase separation driver; in contrast, the disordered 4-6 segment alone cannot phase-separate. Phase separation occurs within a physiologically relevant pH range (4.5-6.5), matching the localized acidification of the cyanobacterial cytoplasm at damaged thylakoid membranes. This directly links membrane stress, pH changes, and IM30 recruitment. Collectively, these findings establish IM30 puncta as bona fide, stress-responsive biomolecular condensates that function as rapid stress sensors and effectors, providing a mechanistic framework for phase separation and condensate formation by bacterial ESCRT-III proteins during environmental adaptation.

biochemistry↗

bioRxiv2023

Protein-specific crowding accelerates aging in phase-separated droplets

Crowding agents, such as polyethylene glycol (PEG, are often used to mimic the cellular cytoplasm in protein assembly studies. Despite the perception that crowding agents have an inert nature, recent work has shown they are not bystanders while proteins interact. Here, we explore the diverse effects of PEG on the phase separation and maturation of proteins. We use two proteins, the FG domain of Nup98 and bovine serum albumin (BSA), which represent an intrinsically disordered protein and a protein with well-established secondary structure, respectively. PEG expedites the maturation of Nup98, enhancing denser protein packing and fortifying hydrophobic interactions which hasten beta-sheet formation and subsequent droplet gelation. In contrast for BSA, PEG appears to enhance droplet stability and limits available solvent for the protein solubilization, without inducing significant changes to the secondary structure, pointing towards a significantly different behavior of the crowding agent. Interestingly, we detect almost no presence of PEG in Nup droplets whereas PEG is detectable within BSA droplets. Our findings demonstrate a nuanced interplay between crowding agents and proteins. PEG can accelerate protein maturation in LLPS systems but its partitioning and effect on protein structure in droplets is protein specific. This suggests that crowding phenomena are specific to each protein-crowding agent pair.

biophysics↗

bioRxiv2023

Structural adaptability and surface activity oftardigrade-inspired peptides

Tardigrades are unique micro-animals that withstand harsh conditions, such as extreme temperatures and desiccation. Recently, it was found that specific cytoprotective proteins are essential for ensuring this high environmental tolerance. In particular, cytoplasmic abundant heat soluble (CAHS) proteins, which are intrinsically disordered, adopt more ordered conformations upon desiccation, and are involved in the vitrification of the cytoplasm. The design and synthesis short peptides capable of mimicking the structural behavior (and thus the cytoprotective properties) of CAHS proteins would be beneficial for potential biomedical applications, including the development of novel heat-resistant preservatives for sensitive drug formulations. As a first step in this direction, we selected several model peptides of varying lengths derived from the conserved CAHS motifs 1 and 2, which are part of the intrinsically disordered CAHS-c2 region. We then studied their structures using circular dichroism and linear and two-dimensional infrared spectroscopy in the presence of the desolvating agent TFE (2,2,2-trifluoroethanol), which mimics desiccation. We found that the CAHS model peptides are mostly disordered at 0% TFE (a result that we confirmed by molecular dynamics simulations), but adopt a more -helical structure upon the addition of the desolvating agent, similar to what is observed for full CAHS proteins. Additionally, we employed sum frequency generation to investigate the surface activity of the peptides at the air/water interface to mimic a partial dehydration effect. Interestingly, all model peptides are surface active and also adopt a helical structure at the air/water interface. Thus, the selected sequences represent promising model peptides that show similarities in the physicochemical behavior to full CAHS proteins. Our results also suggest that arginine might be a crucial element in defining the strong propensity of these peptides to adopt a helical structure. In the future, the use CAHS model peptides to design new synthetic peptide-based materials could make it possible to mimic and exploit the cytoprotective properties of naturally occurring tardigrade proteins. SIGNIFICANCETardigrades are micro-animals that can survive extreme conditions such as desiccation and high temperatures. Recent work has shown that this capability is related to the presence of specific proteins that can remodel in order to protect the organisms cells. Mimicking this behavior using small peptides that preserve the structural properties of the full proteins is highly desirable in potential biomedical applications, such as the storage of heat-sensitive drugs. Here, we study the structural properties of model peptides derived from the conserved region of cytoplastic tardigrade proteins, and show that these peptides preserve some of the conformational behavior of the full protein under drying conditions. These peptides can therefore be used as a starting point for the design of synthetic model systems based on tardigrade-inspired peptides for tailored applications.

biochemistry↗