Biology subjects

Woutersen, S.

Publications and source records attributed to Woutersen, S..

7 recordsLinked to original sources

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↗

bioRxiv2023

Elucidating the role of water in collagen self assembly by isotopically modulating collagen hydration

Water is known to play an important role in collagen self assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H2O/D2O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self assembly. We dissolve collagen in H2O and D2O, and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D2O than in H2O, and collagen in D2O self assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus compared to H2O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D2O is less hydrated than in H2O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions, which appear to be crucial in determining the acceleration of the initial nucleation rate. These results show that water acts as a mediator between collagen monomers, by moderating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self assembly.

biophysics↗

bioRxiv2023

Labile assembly of a tardigrade protein induces biostasis

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins that form gels. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of gelation for CAHS D similar to that of intermediate filaments. We show that gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fiber-like condensates during osmotic stress. Condensation of CAHS D improves survival of osmotically shocked cells through at least two mechanisms: reduction of cell volume change and reduction of metabolic activity. Importantly, condensation of CAHS D is reversible and metabolic rates return to control levels after CAHS condensates are resolved. This work provides insights into how tardigrades induce biostasis through the self-assembly of CAHS gels.

physiology↗

bioRxiv2022

Rheo-2DIR spectroscopy reveals strain-induced hydrogen-bond redistribution in polyurethane

The remarkable elastic properties of polymers are ultimately due to their molecular structure, but the relation between the macroscopic and molecular properties is often difficult to establish, in particular for (bio)polymers that contain hydrogen bonds, which can easily rearrange upon mechanical deformation. Here we show that two-dimensional infrared spectroscopy on polymer films in a miniature stress tester sheds new light on how the hydrogen-bond structure of a polymer is related to its visco-elastic response. We study thermoplastic polyurethane, a block copolymer consisting of hard segments of hydrogen-bonded urethane groups embedded in a soft matrix of polyether chains. The conventional infrared spectrum shows that upon deformation, the number of hydrogen bonds increases, a process that is largely reversible. However, the 2DIR spectrum reveals that the distribution hydrogen-bond strengths becomes slightly narrower after a deformation cycle, due to the disruption of weak hydrogen bonds, an effect that could explain the strain-cycle induced softening (Mullins effect) of polyurethane. These results show how rheo-2DIR spectroscopy can bridge the gap between the molecular structure and the macroscopic elastic properties of (bio)polymers.

biochemistry↗

bioRxiv2022

In situ identification of secondary structures in unpurified Bombyx mori silk fibrils using polarized two-dimensional infrared spectroscopy

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behaviour of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the non-invasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools, because the absorption bands of the amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk filks cast from Bombyx mori silk feedstock. Without any additional analysis, such as peak fitting, we find that the initial effect of hydration is an increase of the random-coil content at the expense of the -helix content, while the {beta}-sheet content is unchanged, and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.

biophysics↗

bioRxiv2021

The giant Staphylococcus epidermidis surface protein Embp binds selectively to fibrillated fibronectin through a Velcro-like mechanism

Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, Embp, which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multi-valent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether. Graphical abstractFibronectin exists in two different conformations in the body. It circulates in the bodily fluids in globular conformation, however, it become fibrillated once adsorbed to an implant surface. S. epidermidis possess a giant 1 MDa receptor known as Embp bind specifically to fibrillated Fn but not to the globular Fn. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/446437v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1999895org.highwire.dtl.DTLVardef@f0717corg.highwire.dtl.DTLVardef@1aa8773org.highwire.dtl.DTLVardef@a2991b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

The Ice Nucleating Protein InaZ is Activated by Low Temperature

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Utilizing specialized ice-nucleating proteins (INPros), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds and may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INPro-induced freezing have remained largely elusive. In the present study, we investigated the folding and the structural basis for interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae strain R10.79. Using vibrational sum-frequency generation and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a {beta}-helical structure in solution and at water surfaces. In this configuration, hydrogen bonding between INPros and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with spectral calculations and molecular-dynamics simulations shows that the INPro reorients at lower temperatures. We suggest that the reorientation can enhance order-inducing water interactions and, thereby, the effectiveness of ice nucleation by InaZ.

biophysics↗