Biology subjects

de Vries, R.

Publications and source records attributed to de Vries, R..

17 recordsLinked to original sources

bioRxiv2026

Surface-stabilized sub-micron condensates for compartmentalizing synthetic cells and enhanced enzyme kinetics

Living cells harbor numerous membraneless organelles (MOs), which are dynamic protein/nucleic acid assemblies responding to specific physicochemical triggers. Liquid-liquid phase separation (LLPS) plays a crucial role in their formation and activity. Inspired by the natural MOs that maintain their individual identities, this work presents a bioengineering strategy to generate LLPS-driven, isochemical MO populations using surfactant-like peptides that stabilize the MO interface. The result is highly stable, monodispersed, sub-{micro}m-sized MO populations, which are not only capable of compartmentalizing synthetic cells but also provide superior environments for enzymatic reactions. This is achieved using pH-responsive elastin-like polypeptides (PREs) as MOs and formulating an amphiphilic PRE-based peptide to stabilize the MO interface. Relative abundance of the surface-active peptide, as well as the rate of pH change, allows direct control over the MO size. Encapsulating these components within synthetic vesicles using a microfluidic platform leads to on-demand multi-compartmentalization via an external pH trigger. Lastly, a functional consequence of the acute size control is shown through a phosphatase reaction, where the highest reaction rate is observed in size-controlled MOs when compared to dilute environments and surfactant-free MOs. The presented strategy provides a new avenue for designing programmable MOs and thus achieve functional compartmentalization within synthetic cells.

bioengineering↗

bioRxiv2026

Hydrophobic and lipid-mediated gating mechanism revealed by low-conductance MthK mutants

Large-conductance calcium (Ca2+)-activated potassium (K+) channels are involved in several essential cellular processes. This requires the ability to alternate between conductive and non-conductive states, yet the molecular mechanism of their gating remains incompletely understood. Bundle-crossing, selectivity filter, and hydrophobic gating are some of the mechanisms previously proposed for these channels, including the archaeal Ca2+-activated K+ channel MthK. Here, we took advantage of two MthK mutants that show dramatically-reduced conductance to gain insights into how the channels gate at the molecular level by combining cryo-electron microscopy, functional assays, and extensive molecular dynamics simulations. Functional and computational analyses reveal distinct mechanisms of reduced conductance between the two mutants. One mutant, where a glutamate residing at the intracellular mouth of the pore was replaced by an alanine (E92A) reduced ion density in the cavity and dramatically accelerated spontaneous channel closure. The second mutant, where an alanine residing in the channels aqueous cavity below the selectivity filter was replaced by a phenylalanine (A88F), introduced a hydrophobic constriction that partially dehydrates the cavity, imposing a barrier to ion permeation. The cryo-EM structures of these mutants, virtually identical to those of WT MthK, were used as models to investigate gating using molecular dynamics (MD) simulations. Long-timescale simulations captured complete open-to-closed transitions in both mutants, uncovering a dehydrated intermediate state and a tightly coupled lipid-entry mechanism that drove final pore closure. Our results show that hydrophobicity and lipid interactions are central determinants of MthK gating.

biophysics↗

bioRxiv2026

Emergence and antigenic characterisation of influenza A(H3N2) viruses with hemagglutinin substitutions N158K and K189R during the 2024/25 influenza season

BackgroundSeasonal human influenza viruses can escape from antibody-mediated neutralization when amino acid changes occur in the hemagglutinin protein. Routine surveillance identified circulation of an A(H3N2) virus variant in the Netherlands with amino acid substitutions at hemagglutinin positions 158 and 189. These amino acid positions were previously responsible for antigenic change of influenza A(H3N2) viruses and potentially lead to escape of this variant from vaccine-mediated immunity. AimTo characterize the emergence and antigenic properties of N158K and K189R double substitution virus variants. MethodsWe analyzed the geographical and temporal dynamics of the double-substitution variant using a phylogeographic approach and used hemagglutination inhibition assays and antigenic cartography methods to map its antigenic properties. ResultsA(H3N2) viruses carrying K189R were first detected in Guatemala in June 2024, before subsequently gaining the N158K substitution, which was intially detected in Colombia in November 2024, followed by detection in the Netherlands in December 2024. However, detections within Europe remained almost entirely confined to the Netherlands. The proportion of viruses carrying the N158K and K189R substitutions increased to 16% - 24% per collection week of sequenced Dutch viruses during the peak of the epidemic of the 2024-2025 respiratory season. Antigenic characterization of viruses with N158K and K189R substitutions indicated that these are antigenically distinct from the A(H3N2) components of 2025-2026 Northern Hemisphere vaccines, showing 8-192-fold reduction in hemagglutination inhibition titers with antisera against the vaccine strain compared to antisera against the homologous virus. ConclusionsInfluenza A(H3N2) viruses with N158K and K189R escaped recognition by antibodies raised against the 2024-2025 and 2025/2026 Northern Hemipshere vaccine strains in hemagglutination inhibition assays. These variants circulated widely in the Netherlands during the 2024-2025 influenza season, raising concerns about reduced vaccine-mediated protection if such variants would spread more broadly during 2025-2026 Northern Hemipshere season.

microbiology↗

bioRxiv2025

Atomistic Mechanism of Calcium-Mediated Inward Rectification of the MthK Potassium Channel by Solid-State NMR and MD Simulations

Inward rectification is a fundamental but poorly understood phenomenon in potassium channel physiology. Despite its physiological importance, the exact mechanism has remained elusive. In this work, we uncover a previously unrecognized calcium-mediated gating mechanism in the MthK potassium channel that sheds new light on this essential process. By combining state-of-the-art proton-detected solid-state NMR spectroscopy with atomistic molecular dynamics simulations, we reveal that divalent calcium ions bind to a novel site just below the selectivity filter, physically obstructing the outward flow of potassium ions whereas inward flow is still possible - analogous to a molecular ball check valve. Secondly, the binding of Ca2+ to the newly identified site leads to stabilization of the selectivity filter and allows us to directly observe ion-ion interactions in the filter. These results offer direct experimental support for the long-debated "direct knock-on" mechanism, in which potassium ions move through the filter, without water co-transport.

biophysics↗

bioRxiv2025

Inhibition of ice recrystallization with designed twistless helical repeat proteins

Given the repetitive structure of crystalline ice, its unsurprising that highly active ice-binding proteins, often with beta-roll structures, also have repeating motifs. Here, we introduce a de novo designed family of ice-binding twistless alpha-helical repeat (iTHR) proteins. Each iTHR protein comprises two planar layers of parallel alpha-helices connected by loops--a structural topology not seen in native ice-binding proteins. The ice-binding helices feature an ordered array of TXXXAXXXAXX motifs, spaced to match the pyramidal {201} and secondary prism plane {110} ice lattice facets, and engineered with a 98.2{degrees} turn angle per residue to orient threonines uniformly towards the ice surface. iTHR proteins show high solubility, thermostability, and produce varied ice crystal morphologies depending on their intended target facet. Crucially, iTHRs exhibit ice recrystallization inhibition (IRI) at critical concentrations comparable to those of many native globular ice-binding proteins. Extensive site-specific mutagenesis shows that ice-binding activity in iTHR proteins is robust, remaining largely unaffected by changes in chemical composition. Variation in the repeat number reveals a non-monotonic relationship to IRI activity. X-ray crystal structures of two designs confirm the intended orientation of threonines, uniformly pointing toward the ice surface. The iTHR family provides a versatile platform to systematically investigate the complex structure-activity relationships underlying protein-ice interactions.

biophysics↗

bioRxiv2025

Sialic acid-containing glycolipids extend the receptor repertoire of Enterovirus-D68

Enterovirus D68 (EV-D68) emerged as a pathogen of increasing health concern globally, particularly due to its association with outbreaks of severe respiratory diseases and acute flaccid myelitis (AFM) in children. Knowledge regarding the tissue tropism and pathogenesis of EV-D68 within the respiratory tract and central nervous system remains limited, primarily due to an incomplete understanding of the host factors that facilitate EV-D68 entry into host cells. Several cellular receptors involved in EV-D68 infections have been identified, including ICAM-5, sialylated glycoproteins, and heparan sulfate (HS). Here, we investigate the receptor requirement of a panel of EV-D68 strains covering all clades focusing on HS and sialosides utilizing glycan arrays. We found that all EV-D68 strains binding to HS harbour a cell culture adaptative substitution in the structural protein VP1 at position 271 which changes the amino acid into a positive charged one. Glycan array analyses revealed that EV-D68 strains either prefer 2,6-linked sialic acids presented on N-glycans, 2,8 linked sialic acids on gangliosides, or both. Inhibition of glycolipid biosynthesis or multivalent glycolipid mimics confirmed that ganglioside structures serve as entry receptors for certain EV-D68 strains. Lastly, we examined whether EV-D68 strains that bind to HS or glycolipids require different uncoating mechanisms. Bafilomycin A1 minimally affected cell entry of HS-binding EV-D68 strains B2/039 and B2/947 and the ganglioside preferring B1/2013 other viruses were strongly inhibited. Together, we identified that EV-D68 strains can use disialoglycolipids as novel receptors and that different EV-D68 strains show a promiscuous sialic acid binding repertoire.

microbiology↗

bioRxiv2024

Computational redesign of TALE proteins for DNA-templated assembly of protein fibers

Many viral proteins self-assemble into capsid structures, often using their genetic material as a template for assembly. To date, de novo designed capsid-like proteins do not require genetic material as a template for assembly, which can be both an advantage and a disadvantage depending on the use case. Templates are indispensable, for example, in the assembly of linear structures with well-defined lengths. As a first step towards fully de novo designed templated assembly, here we redesign proteins from the Transcription activator-like effector (TALE) family of transcriptional regulators to polymerize on double-stranded DNA (dsDNA) templates. Starting from natural TALE protein sequences, we created idealized repeat proteins with sequence-independent DNA binding properties that cooperatively self-assemble to form linear protein-DNA complexes with template-controlled lengths. We used high-resolution atomic force microscopy (AFM) and cryo electron microscopy (cryo-EM) to characterize the three-dimensional structures of the DNA-protein hybrid complexes. In these structures, a protein filament helically wraps around the dsDNA using a binding mode similar to that of natural TALE proteins. As an example application of these materials, we show the system can be used for repetitive peptide antigen display at precisely controlled repeat distances, and that such immunogens elicit robust antigen-specific antibodies in mice.

biochemistry↗

bioRxiv2024

Charge Scaling in Potassium Channel Simulations: Conductance, Ion Occupancy, Voltage Response, and Selectivity

Potassium (K+) channels are widely distributed in many types of organisms. They combine high efficiency ([~]100 pS) and K+/Na+ selectivity by a conserved selectivity filter (SF). Molecular Dynamics (MD) simulations can provide detailed, atomistic mechanisms of this sophisticated ion permeation. However, currently there are clear inconsistencies between computational predictions and experimental results. Firstly, the ion occupancy of the SF in simulations is lower than expected ([~]2.5 in MD compared to [~]4 in X-ray crystallography). Secondly, in many reported MD simulations of K+ channels, K+ conductance is typically an order of magnitude lower than experimental values. This discrepancy is in part because the force fields used in MD simulations of potassium channels do not account for polarization. One of the proposed solutions is the Electronic Continuum Correction (ECC), a force field modification that scales down formal charges, to introduce the polarization in a mean-field way. When the ECC is used in conjunction with the Charmm36m force field, the simulated K+ conductance increases 13-fold. Following the analysis of ion occupancy states using Hamiltonian Replica Exchange (HRE) simulations, we propose a new parameter set for Amber14sb, that also leads to a similar increase in conductance. These two force fields are then used to compute, for the first time, the full current-voltage (I-V) curves from MD simulations, approaching quantitative agreement with experiments at all voltages. In general, the ECC-enabled simulations are in excellent agreement with experiment, in terms of ion occupancy, conductance, current-voltage response, and K+/Na+ selectivity. Significance StatementPotassium (K+) channels are essential membrane proteins that facilitate the selective conduction of K+ ions while excluding Na+ ions. Incorporation of electronic polarization effects through the Electronic Continuum Correction (ECC) method enables molecular dynamics simulations to reproduce rapid K+ permeation under physiological voltage conditions. In this study, we demonstrate that employing ECC approximated polarization in molecular dynamics simulations allows for the prediction of multiple channel properties: conductance, ion occupancy, voltage response, and selectivity, with unprecedented accuracy. Furthermore, our simulations provide atomistic insights into the asymmetrical current-voltage (I-V) relationship observed in the MthK channel.

biophysics↗

bioRxiv2024

Capture of Human Neuromesodermal and Posterior Neural Tube Axial Stem Cells

The spinal cord, nerves, and skeletal muscles arise from neuromesodermal progenitors (NMPs). We have developed a growth-factor screening strategy, utilizing ES and iPS cells, facilitating the indefinite self-renewal of two types of human axial stem cells (AxSCs), closely resembling mouse NMPs (NM-AxSCs) and posterior neural tube progenitors (N-AxSCs). Under specific regimens-- Wnt/CHIR99021, FGF2, and TGF-{beta} inhibitor SB431542 (CFS) and excluding FGF2 (CS), respectively--these AxSCs self-renew and sustain telomeres. Single cell transcriptomics and proteomics have revealed expression of posterior growth-zone and dorsoventral neural tube markers in NM-AxSCs, and correspondingly, differentiation to a wide spectrum of neural tube neurons and myocytes. N-AxSCs rapidly matured into dorsal sensory subsets and neural crest. Crucially, neither AxSC type produces teratomas, and analogous mouse NM-AxSCs integrated successfully into the neural tube and somites. Capturing of AxSCs from patient and GMP ES / iPS cells without transgenesis unveils ontogeny and promises modeling and therapy in neuropathies.

developmental biology↗

bioRxiv2023

Curvature-induced lipid sorting beyond the critical packing parameter

Over the last few decades, the spatial organization of biomembranes has received a lot of attention. With the fluid mosaic model, Singer and Nicolson proposed that biomembranes behave as two-dimensional fluids, allowing proteins and lipids to diffuse freely and thus react to external stimuli. Biological functionality and membrane organization are intricately linked to each other, however, how these organizations emerge is yet to be established. Here we use coarse-grained molecular dynamics simulations to explore the relationship between membrane shape and lateral organization at the nanoscale for a broad range of lipid mixtures. Our results illustrate that membrane curvature and lateral organization are intimately linked at the nanoscale and the relationship is much more complicated compared to the traditional views, in particular the lipid shape concept. Furthermore, we show that large membrane curvature can disrupt phase separation, line tension, and interleaflet coupling leading to non-intuitive lipid sorting. Our findings highlight how curvature-driven sorting can alter the phase behavior of lipid mixtures, which could be crucial for cellular functionality.

biophysics↗

bioRxiv2023

Rapid and automated design of two-component protein nanomaterials using ProteinMPNN

The design of novel protein-protein interfaces using physics-based design methods such as Rosetta requires substantial computational resources and manual refinement by expert structural biologists. A new generation of deep learning methods promises to simplify protein-protein interface design and enable its application to a wide variety of problems by researchers from various scientific disciplines. Here we test the ability of a deep learning method for protein sequence design, ProteinMPNN, to design two-component tetrahedral protein nanomaterials and benchmark its performance against Rosetta. ProteinMPNN had a similar success rate to Rosetta, yielding 13 new experimentally confirmed assemblies, but required orders of magnitude less computation and no manual refinement. The interfaces designed by ProteinMPNN were substantially more polar than those designed by Rosetta, which facilitated in vitro assembly of the designed nanomaterials from independently purified components. Crystal structures of several of the assemblies confirmed the accuracy of the design method at high resolution. Our results showcase the potential of deep learning-based methods to unlock the widespread application of designed protein-protein interfaces and self-assembling protein nanomaterials in biotechnology.

biochemistry↗

bioRxiv2022

Auditory accessory stimulus boosts pupil-linked arousal and reduces choice bias

AO_SCPLOWBSTRACTC_SCPLOWRecent work indicates that pupil-linked phasic arousal signals reduce the impact of prior expectations and biases on decision formation. It has remained unclear whether phasic arousal (i) causes the bias reduction, if (ii) choosing against ones bias causes phasic arousal, or if (iii) a third variable is driving both. Here, using an auditory accessory stimulus, we found evidence for the first scenario: on accessory stimulus vs normal trials, pupil-linked phasic arousal was robustly elevated and choice bias was reduced. With computational modeling of behavior, we established that the bias reduction was not due to a change in response caution (i.e., speed-accuracy tradeoff), but due to a change in a bias in the accumulation of evidence leading up to a choice. Thus, pupil-linked phasic arousal shapes choice behavior.

neuroscience↗

bioRxiv2022

Adipose-derived stromal cells preserve pancreatic islet function in a transplantable 3D bioprinted scaffold

Intra-portal islet transplantation is the method of choice for treatment of insulin dependent type 1 diabetes, but its outcome is hindered by limited islet survival due to the immunological and metabolic stress post transplantation. Adipose-derived stromal cells (ASCs) promise to improve significantly the islet micro-environment but an efficient long-term delivery method has not been achieved. We therefore explore the potential of generating ASC enriched islet transplant structure by 3D bioprinting. Here, we fabricate a double-layered 3D bioprinted scaffold for islets and ASCs by using alginate-nanofibrillated cellulose bioink. We demonstrate the diffusion properties of the scaffold and report that human ASCs increase the islet viability, preserve the endocrine function, and reduce pro-inflammatory cytokines secretion in vitro. Intraperitoneal implantation of the ASCs and islets in 3D bioprinted scaffold improve the long-term function of islets in diabetic mice. Our data reveals an important role for ASCs on the islet micro-environment. We suggest a novel cell therapy approach of ASCs combined with islets in a 3D structure with a potential for clinical beta cell replacement therapies at extrahepatic sites.

cell biology↗

bioRxiv2020

N-glycolylneuraminic acid binding of avian H7 influenza A viruses

Influenza A viruses initiate infection by binding to glycans with terminal sialic acids present on the cell surface. Hosts of influenza A viruses variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in the majority of mammals including horses, pigs, and mice, but is absent in humans, ferrets, and birds. Intriguingly, the only known naturally occurring influenza A viruses that exclusively bind NeuGc are the extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with its NeuGc ligand and observed a high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs. We found that mutation A135E is key for binding 2,3-linked NeuGc but does not abolish NeuAc binding. Interestingly, additional mutations S128T, I130V, or a combination of T189A and K193R, converted from NeuAc to NeuGc specificity as determined by glycan microarrays. However, specific binding to NeuGc-terminal glycans on our glycan array did not always correspond with full NeuGc specificity on chicken and equine erythrocytes and tracheal epithelium sections. Phylogenetic analysis of avian and equine H7 HAs that investigated the amino acids at positions 128, 130, 135, 189, and 193 reveals a clear distinction between equine and avian residues. The highest variability in amino acids (four different residues) is observed at key position 135, of which only the equine glutamic acid leads to binding of NeuGc. The results demonstrate that avian H7 viruses, although genetically distinct from equine H7 viruses, can bind NeuGc after the introduction of two to three mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. Author summaryInfluenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A virus, these keys (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority binds NeuGc. NeuGc is present in species like horses, pigs, and mice, but not in humans, ferrets, and birds. Therefore, NeuGc binding could be a determinant of an Influenza A virus species barrier. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

molecular biology↗

bioRxiv2020

SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

microbiology↗

bioRxiv2019

Oxidative stress in alpha and beta cells as a selection criterion for biocompatible biomaterials

The clinical success of islet transplantation is limited by factors including acute ischemia, stress upon transplantation, and delayed vascularization. Islets experience high levels of oxidative stress due to delayed vascularization after transplantation and this can be further aggravated by their encapsulation and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, PEOT/PBT300 did not induce oxidative stress or reduce viability in MIN6 beta cells, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets. These data indicate that PEOT/PBT300 may be a suitable biomaterial for an islet encapsulation device.

cell biology↗

bioRxiv2019

Real-time assembly of an artificial virus elucidated at the single-particle level

While the structure of a variety of viruses has been resolved at atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues in assembly are the nature of the critical nucleus starting particle growth, the subsequent self-assembly reaction and the manner in which the viral genome is compacted. These issues are difficult to address in bulk approaches and are effectively only accessible by tracking the dynamics of assembly of individual particles in real time, as we show here. With a combination of single-molecule techniques we study the assembly into rod-shaped virus-like particles (VLPs) of artificial capsid polypeptides, de-novo designed previously. Using fluorescence optical tweezers we establish that oligomers that have pre-assembled in solution bind to our DNA template. If the oligomer is smaller than a pentamer, it performs one-dimensional diffusion along the DNA, but pentamers and larger oligomers are essentially immobile and nucleate VLP growth. Next, using real-time multiplexed acoustic force spectroscopy, we show that DNA is compacted in regular steps during VLP growth. These steps, of [~]30 nm of DNA contour length, fit with a DNA packaging mechanism based on helical wrapping of the DNA around the central protein core of the VLP. By revealing how real-time, single particle tracking of VLP assembly lays bare nucleation and growth principles, our work opens the doors to a new fundamental understanding of the complex assembly pathways of natural virus particles.

biophysics↗