Posters

Download list of posters here. Abstracts of semi-confidential posters not shown.

The posters will be located in one end of the conference venue. The poster owners are encouraged to put up their posters as soon as the poster boards are ready (the wall between the Finland and Sweden rooms will be removed).

POSTER NO 1: Alain André

Biosynthesis within bio-inspired engineered artificial condensates

Alain A.M. André, Ankush Garg, P. Nikita K. Rehnberg, Magnus Kjærgaard

Aarhus University

Contact: alain.andre@mbg.au.dk

Biomolecular condensates have emerged as powerful tools for both biological research and biotechnological applications, due to their inherent ability to sequester molecules. This property makes them ideal candidates for use as reaction crucibles in various enzymatic processes. In this study, we engineered a robust and versatile system using synthetic intrinsically disordered repeat proteins (IDPs). These proteins can be precisely tailored to exhibit specific properties such as stability, responsiveness, and molecular affinity, making them excellent platforms for customized enzymatic environments. We focus on characterizing the material properties of these bio-inspired protein condensates to optimize their functionality and stability for potential use in biotechnological applications. As case study we here study the synthesis of naringin from p-coumarate through a three-enzyme cascade. Our findings aim to advance the development of innovative enzymatic crucibles, enhancing the efficiency and specificity of biochemical reactions.

POSTER NO 2: Anamika Biswas

Competitive displacement of lipoprotein lipase is orchestrated by a conserved acidic cluster in GPIHBP1

Anamika Biswas, Kristian K. Kristensen, Samina Arshid, Thomas J.D. Jørgensen, Michael Ploug

Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark

Contact: Anamika.Biswas@finsenlab.dk

The translocation of lipoprotein lipase (LPL) from myocytes or adipocytes to the capillary lumen is critical for intravascular lipolysis and the maintenance of plasma triglyceride homeostasis. Insufficient LPL activity in the capillary lumen results in hypertriglyceridemia. The trans-endothelial transport of LPL is mediated by electrostatic interactions with the intrinsically disordered N-terminal tail of GPIHBP1, which contains two acidic clusters at residues 5–12 and 19–30. This polyanionic tail functions as a molecular switch, facilitating the release of LPL from heparan sulfate proteoglycans (HSPGs) through electrostatic field invasion. In gene-edited mice with a neutralized acidic tail, LPL remains trapped in the sub-endothelial spaces, leading to hypertriglyceridemia. Due to the disordered nature of the tail, the crystal structure of the LPL•GPIHBP1 complex does not elucidate the electrostatic interactions between LPL and GPIHBP1’s acidic tail. In this study, we employed zero-length crosslinking to map the acidic tail of GPIHBP1 onto LPL. Carboxylates at positions 19–30 in GPIHBP1 were found to interact with Lys445, Lys441, Lys414 and Lys407 near the interface of the C- and N-terminal domains of LPL. Modeling these interactions revealed an extensive electrostatic interface encompassing both LPL domains, which accounts for the stabilization of LPL activity and conformation by the acidic tail. Functional assays demonstrated that the acidic cluster at residues 19–30 is crucial for maintaining LPL activity, mitigating ANGPTL4-mediated LPL inactivation, preventing PSCK3-induced LPL cleavage, and facilitating LPL extraction from HSPGs. Our findings provide molecular insights into the electrostatic regulation and compartmentalization of LPL activity, which are essential for intravascular lipolysis and plasma triglyceride homeostasis.

POSTER NO 3: Ankush Garg

Biomolecular condensates exclude oxygen via enhanced protein density

Ankush Garg(1), Christopher Brasnett(2), Siewert J. Marrink(2), Klaus Koren(3), Magnus Kjaergaard (1,4,5)

(1) Department of Molecular Biology and Genetics, Aarhus University, Denmark. (2) Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. (3) Department of Biology, Aarhus University, Denmark. (4) Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark. (5) The Danish Research Center for Translational Neuroscience, Aarhus University, Denmark

Contact: au711045@uni.au.dk

Biomolecular condensates form through the self-assembly of proteins and nucleic acids to create dynamic compartments in cells. By concentrating specific molecules, condensates establish distinct microenvironments that regulate biochemical reactions in time and space. Macromolecules and metabolites partition into condensates depending on their interactions with the macromolecular constituents, however, the partitioning of gases has not been explored. We investigated oxygen partitioning into condensates formed by multiple repeats of intrinsically disordered octapeptide using electrochemical oxygen sensing and phosphorescence lifetime imaging microscopy (PLIM) technique. Both the experiments showed partial exclusion of oxygen from the condensates. We have systematically varied the chain length and hydrophobicity to examine the molecular reason behind oxygen exclusion. The oxygen concentration within condensates increases with increasing chain length of the octapeptide repeats. The dense phase protein concentration drops with chain length indicating anti-correlation between oxygen partitioning and protein concentration. However, we do not see any clear trend with varying hydrophobicity. This suggests that oxygen partitioning is determined by the physical organization of the condensates rather than the chemical properties of the scaffold. Molecular dynamics simulations suggest that oxygen does not form strong and specific interactions with the scaffold and is dynamic on the nanosecond timescale. Biomolecular condensates thus result in variation of oxygen concentrations on micron scale length-scales, which can tune the oxygen concentration available for biochemical reactions within the cell.

POSTER NO 4: Annette Juma Nielsen

From Alanine Scanning to High-Throughput DMS: Characterizing ArcCA’s Low-Affinity Ligand Binding

Annette Juma Nielsen (1), Christian Parsbæk Buch (1), Joseph Matthew Rogers (2) & Kaare Teilum (1)

(1) Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen. (2) Department of Drug Design and Pharmacology, University of Copenhagen.

Contact: annette.juma@bio.ku.dk

The activity-regulated cytoskeleton-associated protein (Arc) functions as a critical interaction hub in neuronal postsynaptic dendrites, essential for synaptic plasticity and memory formation. While the N-lobe of Arc's capsid domain (ArcCA) binds proteins involved in clathrin-mediated endocytosis, its optimal binding motif remains unknown. Here, we used alanine scans to systematically identify key residues in two 12-residue peptides, TARP-γ2 and GluN2A, that influence ArcCA binding affinity, expanding the known binding motif. Building on these insights, we are developing a high-throughput deep mutational scanning (DMS) platform using weak-affinity chromatography (WAC) combined with mRNA display. This approach allows us to study μM-affinity interactions that are otherwise challenging to capture. Once fully developed, this platform aims to provide a powerful tool for examining binding specificity in Arc and other protein interactions, advancing our understanding of weak-affinity ligand binding in complex systems.

POSTER NO 5: Anni Kumari

ANGPTL3/8 is an atypical unfoldase that regulates intravascular lipolysis by catalyzing LPL inhibition

Anni Kumari (a,b), Sanne W.R. Larsenb (c), Signe Bondesenb (c), Yuewei Qian (d), Hao D. Tian (e), Sydney G. Walker (f), Brandon S. J. Davies (f), Alan T. Remaley (e), Stephen G. Young (g,h), Robert J. Konrad (d), Thomas J.D. Jørgensen (c), and Michael Ploug (a,b)

(a) Finsen Laboratory, Copenhagen University Hospital - Rigshospitalet, DK–2200 Copenhagen N, Denmark; (b) Biotechnology Research and Innovation Centre (BRIC), University of Copenhagen, DK-2200 Copenhagen N, Denmark; (c) Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK–5320 Odense M, Denmark; (d) Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 462585, United States; (e) Laboratory of Lipoprotein Metabolism, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20814, United States. (f) Department of Biochemistry and Molecular Biology, University of Iowa, Iowa 52242, United States; (g) Department of Medicine, University of California, Los Angeles, CA 90095, United States; (h) Department of Human Genetics, University of California, Los Angeles, CA 90095, United States.

Contact: anni.kumari@finsenlab.dk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 6: Arriën Symon Rauh

Probing the Interactions in Biomolecular Condensates using Simulations of Double Mutant Cycles

Arriën Symon Rauh, Giulio Tesei, and Kresten Lindorff-Larsen

Structural Biology & NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, DK-2200 Copenhagen N

Contact: arrien.rauh@bio.ku.dk

The relationship between the sequence and and phase separation (PS) behaviour of intrinsically disordered proteins (IDPs) is often interrogated through mutagenesis experiments [1-4]. In these experiments either the interactions of specific amino acid residues, or sequence patterns in general are perturbed. To get a more detailed understanding of how these mutations affect the biomolecular condensates we explore the application of double mutant cycles (DMCs) in molecular dynamics (MD) simulations to quantify the impact of sequence perturbations [5-7]. To this end, we use the effective residue-level coarse-grained MD model CALVADOS [8,9]. Here we present the case of tyrosine-arginine interaction(s) in the hnRNPA1-LCD (A1). We can quantify a contribution to stabilising condensates of A1. However, interpreting the alterations in interaction pattern in the condensate is not straightforward, due to condensate concentration differences.

 

[1] Wang et al. Cell (2018)

[2] Borcherds et al. Curr. Op. in Str. Bio. (2021)

[3] Martin et al. Science (2020)

[4] Bremer et al. Nat.Chem. (2022)

[5] Carter et al. Cell (1984)

[6] Fersht et al. J. Mol. Bio. (1992)

[7] Horovitz Folding and Design (1996)

[8] Tesei et al. PNAS (2021)

[9] Tesei & Lindorff-Larsen Open Res. Eur. (2022)

POSTER NO 7: Azad Farzadfard

The amplification of alpha-synuclein amyloid fibrils is suppressed under fully quiescent conditions

Azad Farzadfard¹, Thomas O. Mason¹, Antonin Kunka¹, Hossein Mohammad-Beigi¹, Kaare Bjerregaard-Andersen², Jonas Folke³, Susana Aznar Kleijn³, Pekka Kallunki², Alexander K. Buell^1*

¹ DTU Bioengineering, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. ² H. Lundbeck A/S, Carl Jacobsens Vej 22, 2500 Copenhagen, Denmark. ³ Research Laboratory for Stereology and Neuroscience, Bispebjerg Hospital, 2400 Copenhagen, Denmark

Contact: seyfa@dtu.dk

Seed amplification assays (SAAs) show high accuracy and sensitivity for the diagnosis of neurodegenerative diseases, however, it faces challenges to accurately quantify fibrils in the currently used multiwell plate format. It is therefore desirable to transfer such assays into a digital format in microemulsion droplets to enable direct quantification of aggregate numbers.

We establish a new set of assay conditions that enable highly efficient seed amplification in plates without any shaking. However, the same set of conditions displayed a very different behavior upon transfer to a microfluidic platform where no amplification was observed. We demonstrate that this is caused by the suppression of all secondary processes that could amplify the seeds in the complete absence of mechanical perturbations inside the microdroplets. We further show that the amplification inside droplets can be achieved by subjecting the microemulsions to high-frequency vibrations using a piezo device.

POSTER NO 8: Beenish Sadaqat

Expression, characterization, and substrate specificity of GH36 -galactosidase from Roseburia hominis

Beenish Sadaqat, Lovisa Majtorp, Simon Birgersson, Henrik Stålbrand

Biochemistry and Structural Biology, Center of Molecular Protein Sciences. Lund, Sweden

Contact: beenish.sadaqat@biochemistry.lu.se

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 9: Bram Mylemans

De novo design of minimal proteins for targeted protein degradation

Bram Mylemans, Amanda Acevedo-Jake, Boguslawa Korona, Laura S. Itzhaki, Andrew J. Wilson, Derek N. Woolfson

School of Chemistry, University of Bristol; School of Chemistry, University of Birmingham; Department of Pharmacology, University of Cambridge; Department of Pharmacology, University of Cambridge;

School of Chemistry, University of Birmingham; School of Chemistry, University of Bristol, Max Planck-Bristol Centre for Minimal Biology, University of Bristol, School of Biochemistry, University of Bristol

Contact: bram.mylemans@bristol.ac.uk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 10: Celia Fricke

Thermodynamic stability of alpha-synuclein fibrils as a determinant of disaggregation by chaperones

Celia Fricke[a], Antonin Kunka[a], Rasmus Krogh Norrild[a], Thi Lieu Dang[b], Anne Wentink[c], Bernd Bukau[b], Alexander K. Buell[a]

[a] DTU BIOENGINEERING, Department of Biotechnology and Biomedicine, [b] Heidelberg University, Zentrum für Molekulare Biologie, [c] Leiden University, Leiden Institute of Chemistry

Contact: celfri@dtu.dk

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder with no current effective treatment options. The aggregation and fibril formation of the amyloidogenic protein alpha-synuclein leads to the death of dopaminergic neurons, which causes typical PD symptoms such as tremors. While the process and the kinetics of amyloid fibril formation by alpha-synuclein and other proteins are well studied, little is known about the stability and disaggregation of existing amyloid fibrils. However, the accumulation of amyloid fibrils in the brain and their ability to produce and catalyze the generation of toxic oligomers emphasizes the necessity of a clear picture of the stability of amyloid fibrils and their persistence towards disaggregation by chaperone systems. Here, we investigated the stability of two alpha-synuclein fibril polymorphs over three months using chemical depolymerization assays with flow-induced dispersion analysis (FIDA, [1]) and found that the stability of both polymorphs increased over time. We furthermore investigated the disaggregation of these polymorphs by the chaperone system HSP70, DNAJB1 and Apg2. Interestingly, disaggregation of the polymorphs was seen at all time points, but the degree of disaggregation decreased with increasing stability of the amyloid fibrils. These findings provide insights into amyloid fibril reversibility from a new perspective which has the potential to be a new target for drug molecules that destabilize amyloid fibrils leading to a facilitated disaggregation.

 

[1] Farzadfard A, Kunka A, Mason TO, Larsen JA, Norrild RK, Dominguez ET, Ray S, Buell AK. Thermodynamic characterization of amyloid polymorphism by microfluidic transient incomplete separation. Chem Sci. 2024 Jan 8;15(7):2528-2544. doi: 10.1039/d3sc05371g.

POSTER NO 11: Daria Gusew

Combining NMR spectroscopy and Molecular Dynamics simulations to study conformational dynamics of proteins and protein-ligand interactions

Daria Gusew, Kresten Lindorff-Larsen

Structural Biology and NMR Laboratory, Lindstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen

Contact: daria.gusew@bio.ku.dk

Understanding the processes that control conformational states and conversion between long-lived states is important as they determine the dynamics of a protein and protein-ligand interactions. In nuclear magnetic resonance (NMR) spectroscopy, the chemical shift provides informational about the structural details of a protein. The exchange between conformational states (chemical exchange) due to fluctuations in chemical shifts can be used to study the dynamics of proteins with NMR experiments. Here, we model chemical exchange dynamics by mapping the dynamics of a particle on a 1-D free energy potential extracted from an explicit-water molecular dynamics (MD) simulation [1] of the L99A variant of T4-Lysozyme. Transitions between conformational states on the free energy surface from numerical integration of the Langevin equation can be used to evaluate time autocorrelation functions associated with NMR observables.

 

[1] Yong Wang, Elena Papaleo, Kresten Lindorff-Larsen (2016) Mapping transiently formed and sparsely populated conformations on a complex energy landscape eLife 5:e17505, https://doi.org/

POSTER NO 12: Emil Thomasen

Interdomain interactions in the nuclear receptor PPARγ from an integrative ensemble model

1. Emil Thomasen, Elisabeth G. K. Thomsen, Milena Lalic, Andreas Prestel, Lukas W. Bauer, Johan G. Olsen, Cy Jeffries, Kresten Lindorff-Larsen, Birthe B. Kragelund

Structural Biology and NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen N, Denmark; REPIN; European Molecular Biology Laboratory (EMBL), Hamburg Unit, Deutsches Elektronen-Synchrotron, Hamburg, Germany

Contact: fe.thomasen@bio.ku.dk

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates adipocyte differentiation, lipid metabolism, and insulin sensitivity. PPARγ is a multidomain protein consisting of a disordered AB domain, a DNA-binding domain (DBD), and a ligand binding domain (LBD). The AB-domain is involved in the recruitment of CBP- and p300-containing coactivators, and plays a gene-specific role in PPARγ-mediated transactivation. Here, we characterize the interplay between the disordered AB domain and the folded domains of PPARγ. We use Bayesian maximum entropy reweighting to fit coarse-grained molecular dynamics simulations to SAXS and NMR relaxation data on full length PPARγ. The resulting ensemble model reveals interactions between the disordered AB domain and the DBD, while R2 relaxation data shows a disruption of the AB-DBD interaction upon DNA binding. Our results suggest that DNA binding displaces the AB domain from the DBD, making the AB domain available for coactivator recruitment.

POSTER NO 13: Eva Smorodina

Structural modeling of antibody variant epitope specificity with complimentary experimental and computational techniques

Eva Smorodina

Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway

Contact: eva.smorodina@medisin.uio.no

Antibodies are key immunotherapeutic biomolecules with a hallmark feature of antigen-specific binding. However, the principles governing the ability of diverse paratopes to bind to the same epitope with comparable affinity and specificity remain largely unexplained. An insufficient understanding of the structural rules behind antibody-antigen binding, due to a lack of experimentally resolved structures, leads to the current inability to characterize antibody variants binding in silico. Here we propose a rule-based antibody design that relies on a thorough understanding of epitope-paratope interactions, in contrast to generative design based on millions of trials and errors. We identified the epitope of five affinity-verified Trastuzumab variants using cryo-EM and position-resolved HDX-MS. Rigid models alone are insufficient for accurate antibody-antigen modeling while molecular dynamics simulations with computational analysis of the complex conformations succeed in replicating and complimenting experimental findings. Structural parameters calculated based on geometry, surface, and biochemical properties were able to distinguish between high and low binders. We highlight the possibilities of AI in antibody and antibody-antigen structure modeling, demonstrating the limitations of various language-based models to predict and understand antibody variants. Overall, our study explains the binding mechanisms of the variant sequences, showing how antibodies with diverse sequences share similar antigen-binding rules.

POSTER NO 14: Fabian Hecker

Hyperpolarized water via UV-generated radicals to study the hydration of proteins

Fabian Hecker, Magnus Karlsson, Kaare Teilum, Mathilde H. Lerche

FH, MK, MHL: HYPERMAG, Technical University of Denmark

KT: University of Copenhagen

Contact: fabhec@dtu.dk

Studying water molecules in and around proteins presents a challenge due to their similarity to the bulk solvent. One powerful method to gain insight into protein hydration is Hydrogen-Deuterium (H/D) exchange, a technique pioneered by the namesake of this symposium.[1] Hyperpolarized water (HyperW), generated through dissolution dynamic nuclear polarization (dDNP), offers an innovative approach by significantly enhancing the sensitivity of NMR for biomolecules under mild conditions.[2] Importantly, HyperW relies intrinsically on H/D exchange to transfer hyperpolarization, making it a novel tool for probing water dynamics with fast and sensitive detection.

 

UV-generated radicals from pyruvic acid are ideal for dDNP, as they are temperature-unstable above 190 K. During dissolution, these radicals decompose into diamagnetic species, creating radical-free, slow-relaxing hyperpolarized solutions.[3] This property is critical to producing reproducible 1H polarizations exceeding 30%, with relaxation times extending up to one minute.

 

We demonstrate the application of UV-generated radicals to produce hyperpolarized water and employ it in 2D 1H-15N correlation experiments using SOFAST-HMQC. These experiments allow us to study hydration dynamics in the chymotrypsin inhibitor 2 (CI2), distinguishing between fast- and slow-exchanging backbone amides by comparing HyperW-enhanced SOFAST-HMQC recorded within 16 seconds of injection to thermally polarized samples. We detect critical CI2 residues with greater sensitivity than standard HSQC experiments, indicating their role in protein function.

 

Literature:

[1] A. Hvidt, K. Linderstrøm-Lang, Biochim. Biophys. Acta 1954, 14, 574-575.

[2] C. Hilty, D. Kurzbach, L. Frydman, Nat. Protoc. 2022, 17, 1621-1657.

[3] A. C. Pinon, A. Capozzi, J. H. Ardenkjær-Larsen, Comm. Chem. 2020, 3, 57.

POSTER NO 15: Fan Cao

A coarse-grained model for disordered and multi-domain proteins

Fan Cao, Sören von Bülow, Giulio Tesei, Kresten Lindorff-Larsen

Structural Biology and NMR Laboratory & the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Contact: fan.cao@bio.ku.dk

Multi-domain proteins (MDPs) have more than one folded domain connected by intrinsically disordered regions (IDRs). Because of their larger size and often substantial dynamics, characterising the conformational ensembles of MDPs by simulations may be difficult. We present a coarse-grained model for MDPs and intrinsically disordered proteins (IDPs) that is both fast and provides an accurate description of the global conformational properties in solution. We use our reparameterized model to simulate phase separation of both IDPs and MDPs, study the effect of florescent tags on phase behaviour of proteins and examine how the stability of folded domains may differ between the dilute and dense phases.

POSTER NO 16: Francisca Pinheiro

Targeting peptide drugs based on the activity state of synapses

Francisca Pinheiro and Magnus Kjærgaard

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, DK-8000; The Danish Research Institute for Translational Neuroscience (DANDRITE), Aarhus University, Aarhus, Denmark, DK-8000; Center for Proteins in Memory – PROMEMO, Danish National Research Foundation, Aarhus, Denmark, DK-8000

Contact: francisca.gpinheiro@mbg.au.dk

The overactivity of excitatory neurotransmission has been implicated in the pathogenesis of several brain diseases, turning the components of glutamatergic synapses into attractive drug targets. Yet, the development of drugs that reduce glutamatergic transmission has been largely hindered by the need to preserve normal brain function. In this project, we will engineer a coiled coil that undergoes phosphorylation-dependent dimerization in the presence of activated CAMKII, a biochemical feature of strong excitatory synapses. Thus far, we have redesigned the homodimeric antiparallel coiled coil APH6 to incorporate the recognition motif of one of the most abundant CAMKII brain isoforms, CAMKIIα. In silico predictions suggest that the redesigned coiled coil is less stable than the wild-type protein, as intended, and that phosphorylation improves its stability. In addition, we aim to develop a tool that allows us to assess antiparallel coiled coil formation in a rapid and effective manner, facilitating the screening of different sequences. Importantly, once we have a peptide that assembles into an antiparallel coiled coil upon CAMKII phosphorylation, we will address whether this occurs in primary neurons after long term potentiation. The creation of CAMKII dimerization modules will open a new avenue for studying the pathogenic mechanisms underlying neurologic disorders and, especially, for the development of drugs that target specific activity states of synapses.

POSTER NO 17: Frederik Friis Theisen

The Dual Identity of Proline: Exploring the Effects of Proline Isomerization in IDP Interactions

Frederik F. Theisen, Andreas Prestel, Nina L. Jacobsen, Oline K. Nyhegn-Eriksen, Johan G. Olsen, Birthe B. Kragelund

Institut de Biologie Structurale, Grenoble, France

Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Denmark

Contact: frederik.theisen@bio.ku.dk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 18: Freia Buus

Phase-Separation of Two Highly Charged Disordered Proteins at the Residue Level Explored by NMR Spectroscopy

Freia Stryhn Buus^1,2, Nicola Galvanetto^3,4, Jacob H. Martinsen^1,2, Andreas Prestel¹, Benjamin Schuler^3,4, Birthe B. Kragelund^1,2.

¹REPIN, ²Structural Biology and NMR Laboratory, The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Denmark, ³Department of of Biochemistry, University of Zurich, Zurich, Switzerland, ⁴Department of Physics, University of Zurich, Zurich, Switzerland.

Contact: freia.buus@bio.ku.dk

The two disordered proteins, linker histone H1.0 (H1) and its nuclear chaperone, prothymosin- (ProT), form an electrostatically driven complex, which is notable for retaining its highly disordered and dynamic nature (1). Both proteins have been described as polyelectrolytes, with H1 highly positively charged (+53) and ProT highly negatively charged (-43). Recent findings have revealed an intriguing facet of this interaction – its capacity to drive the formation of biomolecular condensates. At high protein concentrations and low salt levels, H1 and ProT can phase separate into a protein-depleted dilute phase and a protein-enriched and viscous dense phase. The proteins are about 1000 times more concentrated in the dense phase than in the dilute phase. Interestingly, though highly packed in the dense phase, these disordered proteins remain highly dynamic, with rapidly changing configurations (2). Here we utilize the power of nuclear magnetic resonance (NMR) spectroscopy to explore this phenomenon at the residue level. The challenge is to accurately separate the two phases for analyses by NMR spectroscopy. We have used a set of approaches, including sedimentation of the formed liquid droplets into a biphasic sample and the isolation of the dilute phase, as well as using a few percentages of agarose to stabilize the liquid droplets. The latter method results in an across-sample homogeneity, enabling the simultaneous detection of proteins in dilute and dense phases (3). With these experimental setups, changes at a residue-specific level among proteins in the dilute and dense phases can be simultaneously probed. Thus, our work establishes a set of approaches for employing NMR spectroscopy to investigate highly charged intrinsically disordered proteins within biomolecular condensates at a high resolution. This holds a valuable potential for advancing our understanding of how intrinsic disorder and polyelectrolyte properties, as exemplified by systems like H1 and ProT, contribute to the formation and regulation of condensate formation.

 

References: (1) Borgia A, Borgia MB, Bugge K, et al. Extreme disorder in an ultrahigh-affinity protein complex. Nature. 2018;555(7694):61-66. (2) Galvanetto N, Ivanović MT, Chowdhury A, et al. Extreme dynamics in a biomolecular condensate. Nature. 2023;619(7971):876-883. (3) Emmanouilidis L, Esteban-Hofer L, Damberger FF, et al. NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation. Nat Chem Biol. 2021;17(5):608-614.

POSTER NO 19: Giulio Tesei

Conformational ensembles of the human intrinsically disordered proteome

Giulio Tesei, Anna Ida Trolle, Nicolas Jonsson, Johannes Betz, Frederik E. Knudsen, Francesco Pesce, Kristoffer E. Johansson, and Kresten Lindorff-Larsen

University of Copenhagen

Contact: giulio.tesei@bio.ku.dk

Intrinsically disordered regions (IDRs) comprise about one third of the human proteome and play key roles in biological processes. While lacking fixed 3D structures, IDRs adopt diverse conformational ensembles determined by their amino acid sequences, which are not yet fully captured by current structure prediction methods. Through simulations using the CALVADOS model and bioinformatics, we built a proteome-wide database of IDR ensembles, and explored relationships between sequence, IDR conformational ensemble, and protein function, as well as the evolutionary conservation of IDR conformational properties.

POSTER NO 20: Jens Nicolai Decker

Pioneer factor Sox2 modulates nucleosome architecture

Jens Nicolai Decker, Sveinn Bjarnason, Jordan McIvor, Davide Mercadante, Pétur O. Heiðarsson 

Structural Biology and NMR Laboratory, University of Copenhagen

Contact: jens.decker@bio.ku.dk

Pioneer transcription factors (pTFs) activate genes by binding to and remodeling condensed chromatin, enabling access to otherwise inaccessible genomic regions. This remodeling is crucial for cellular reprogramming by facilitating access to key pluripotency genes. The pTF Sox2 is essential for stem cell pluripotency and driving cellular reprogramming by reshaping the chromatin landscape. A major challenge in understanding the mechanistic details of chromatin remodeling, is the currently limited structural description of Sox2. While the DNA binding domain (DBD) of Sox2 is well studied, the intrinsically disordered C-terminal region (C-IDR) is less understood, despite its critical role in transcriptional activation and pioneer function.
To explore how full-length Sox2 remodels DNA and nucleosomes, we use advanced techniques such as single molecule Förster Resonance Energy Transfer and nuclear Magnetic Resonance spectroscopy combined with molecular dynamics simulations. We studied Sox2 in complex with both free dsDNA as well as nucleosomes reconstituted either with strongly positioning or endogenous DNA sequence.

Upon DNA and nucleosome binding, the C-IDR undergoes substantial structural rearrangements that includes a redistribution of the relative accessibility of its two activation domains. Interestingly, we observed that in complex with nucleosomes, rapid sub millisecond dynamics in the C-IDR are reduced compared to when bound to DNA, suggesting a specific interaction between C-IDR and the histone octamer. Notably, high affinity binding was consistent across different Sox2 binding sites when placed in the nucleosome core structure but introducing additional linker DNA increased binding affinity ~65-fold, suggesting that Sox2 preferably binds to accessible linker regions.

By revealing how the Sox2 C-IDR contributes to nucleosome interactions and remodeling, our results advance our understanding of pioneer transcription factors and lays the groundwork for refining cellular reprogramming strategies.

POSTER NO 21: Ida Kjærsgaard Grene

De-novo protein design for neuroscience: Homology and specificity

Ida Kjærsgaard Grene1,2,3 and Magnus Kjærgaard1,2,3

1. Department of Molecular Biology and Genetics, Aarhus University
2. Danish Research Institute of Translational Neuroscience (DANDRITE)
3. Center for Proteins in Memory (PROMEMO)

Contact: ikg@mbg.au.dk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 22: Ida Marie Vedel

Unveiling the competitive interactions of Dab2 and Eps15 during initiation of clathrin mediated endocytosis

Ida M Vedel, Andromachi Papagiannoula, Kathrin Motzny, Sigrid Milles

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

Contact: vedel@fmp-berlin.de

Disabled-2 (Dab2) is a clathrin associated sorting protein (CLASP), which functions as an adaptor protein, particularly important in the early stages of clathrin mediated endocytosis (CME), one of the major uptake pathways of eukaryotic cells. The CME cargo is often transmembrane receptors and consequently CME, and thus also Dab2, play vital roles in cell signaling. In the CME process, Dab2 and other CLASPs, which all contain long intrinsically disordered regions (IDRs), form an intricate interaction network along with the major adaptor protein AP-2. This network functions to recruit necessary proteins and to bridge between clathrin, the membrane, and the cargo, in the end resulting in the clathrin coated vesicle to be internalized. While AP-2 is usually required for successful CME, Dab2 is a special CLASP as it has been shown to mediate CME independently of AP-2. The IDR of Dab2 contains multiple, and repetitive, small amino acid motifs that are expected binding sites for clathrin, AP-2, and other CLASPs, in addition to multiple (putative) phosphorylation sites. We use solution NMR to study Dab2’s conformational dynamics, its interaction with CME partner proteins, and the effects of differential phosphorylations, which will shed light on Dab2’s function in both the AP-2 dependent and independent CME protein network. Interaction studies with EH domains of the partner CLASP Eps15 show that Dab2 partially competes with intramolecular interactions within Eps15 revealing a highly complex and competitive interaction network, likely to have implications for liquid-liquid phase separation in endocytosis.

POSTER NO 23: Jan Stanislaw Nowak

Fast Track to Functional Binders:  Screening De Novo Proteins with FIDA

Jan Stanislaw Nowak, Elena Zueva, Francisca Pinheiro, Magnus Kjærgaard

Department of Molecular Biology & Genetics, Aarhus University

Contact: jsn@mbg.au.dk

Recent advances in machine learning and computational tools have significantly advanced the field of de novo protein design, enabling researchers to rapidly and affordably develop protein binding partners. As a result, the output rate of promising candidates has increased, making experimental validation the primary limiting factor in terms of time, labor, and capacity. We are exploring new experimental screening methods for de novo protein binders using flow-induced dispersion analysis (FIDA). This technique allows us to assess the stability, folding, and binding affinity of candidate proteins with microliter sample volumes. Additionally, we screen for binding, stability, and expression directly in lysates from binder-expressing bacteria, a method that reduces waste by minimizing the production of non-functional binders.

POSTER NO 24: Jesper Elmsted Dreier

Small molecule mediated inhibition of α-synuclein amyloid fibril formation

Jesper E. Dreier¹, Alisdair Stevenson², Thomas C.T. Michaels² and Céline Galvagnion¹

¹ Department of drug design and pharmacology, University of Copenhagen

² Department of Biology, Institute of Biochemistry, ETH Zurich

Contact: jesper.dreier@sund.ku.dk

Parkinson’s disease (PD) is characterized by a loss of dopaminergic neurons as well as the deposition of protein-lipid inclusions called Lewy bodies (LB) whose main constituent is the pre-synaptic protein α-synuclein(αS). αS is involved in synaptic plasticity via its binding to vesicles; however, this protein-membrane interaction can also lead to the formation of amyloid fibrils resembling those found in LB. Many findings suggest that anionic lipids not only initiate αS aggregation but also incorporates into the amyloid fibrils.

Mutations in the gene GBA1, which codes for the enzyme glucocerebrosidase (GCase), is the most important genetic risk factor for PD and is associated with lysosomal disorders, lipid and αS accumulation. A small molecule, ambroxol (ABX) has been found to restore wild type function in the mutant variants and consequently restoring lipid and αS levels to normal, by acting as a molecular chaperone for Gcase. Despite the fact that Abx is currently in phase III clinical trials for PD patients with GBA mutations, the effect of ABX itself on the aggregation of αS remains unknown.

 

Using a combination of biophysical methods, we discovered that ABX inhibits the lipid-induced aggregation of αS and can displace the protein from the membrane.  Moreover, kinetic analyses of Thioflavin-T aggregation curves provided the mechanism of inhibition and showed that ABX not only displace αS from the membrane but also slow down the formation of oligomers.

 

These results are highly relevant, not only because they provide a pipeline that could be useful in testing other potential drugs against protein aggregation, but also because they show that a small molecule capable of reverting PD related lipid changes can also directly impact αS by inhibiting its aggregation. This finding is the first evidence for a small molecule to have dual mode of action, acting on both the lipid metabolism and αS aggregation.

POSTER NO 25: Jing Zhao

Discovery of de novo molecular glues

Jing Zhao, Joseph Rogers

Department of Drug design and Pharmacology, University of Copenhagen, Denmark

Contact: jing.zhao@sund.ku.dk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 26: Joel Chubb

: RASSCoL: Simplifying Computational Design of Small-Molecule Binding Pockets

Joel J. Chubb, Katazyna Ożga, Rokas Petrėnas, Derek N. Woolfson

School of Chemistry, University of Bristol

Contact: joel.chubb@bristol.ac.uk

Designing proteins with precise small-molecule binding capabilities is a current challenge in protein design, with implications for drug discovery, biosensing, and synthetic biology. Current methods often involve complex, time-consuming approaches requiring levels of in vitro screening not widely accessible and that may not yield the desired specificity or affinity.

 

To address these limitations, we present RASSCoL (Rapid Assessment of Size and Shape Complementarity of Ligands). RASSCoL is a two-stage computational approach that integrates volume displacement with fast computational screening to efficiently design small-molecule binding pockets in de novo proteins. In the first stage, we employ exhaustive sampling and docking of potential sequence candidates generated using a volume displacement technique that reshapes binding pockets to enhance their complementarity with target molecules. This method allows us to rapidly generate a pool of candidates, each tailored to accommodate specific small molecules. The second stage involves accelerated molecular dynamics simulations to evaluate and refine these candidates, ensuring both accuracy and binding affinity.

 

Our approach has successfully produced binders with nanomolar (nM) to micromolar (μM) affinities for a selection of fluorescent molecules, demonstrating specificity and selectivity. Furthermore, we have engineered dual binders from these designs capable of energy transfer between molecules, illustrating the functional versatility of our designs. Building on these successes, we are now extending our pipeline to more complex targets, including pharmacologically relevant compounds. This highlights the potential of our design pipeline to contribute to the development advanced biosensors and function assays. Our streamlined approach represents an advance in the field of protein design, offering a new tool for the rational design of protein-based binders with high specificity and functionality.

POSTER NO 27: Joseph

De novo peptides and proteins to modulate protein disorder

Fabian Hink, Jesper E. Dreier, Joseph Watson, Estella Newcombe, Birthe B. Kragelund, Céline Galvagnion, David Baker, Joseph M. Rogers

University of Copenhagen

Contact: joseph.rogers@sund.ku.dk

Defects in protein folding, folding-upon-binding, and disorder are the root cause of many human disorders, underlying the importance of these key biophysical processes in the functioning of the cell. Molecules able to modulate these processes would be valuable as research tools, could form the basis of future therapeutics to correct these defects, or both. Yet discovery of such molecules presents a significant challenge to existing drug/ligand modalities and classical screening methods. Here, we present two promising modalities to influence protein biophysical behavior, de novo cyclic peptides and de novo microproteins, and the experimental and computational breakthroughs that have enabled their discovery. We report two example of these modalities modulating particular protein biophysical behaviour: de novo microproteins that can influence folding-upon-binding of a disordered peptide and de novo cyclic peptides that can bind a modulate aggregation of a model disease protein.

POSTER NO 28: Julian Beck

Scaffolding a Kemp Eliminase Activity into an idealized de novo TIM barrel

Julian Beck, Emily Freund, Ben Smith, Niayesh Zarifi, Roberto Chica, Birte Höcker

University of Bayreuth

Contact: julian.beck@uni-bayreuth.de

One milestone in protein design was the design of the first de novo TIM barrel. However, the introduction of functions into de novo TIM barrels is challenging due to their idealized scaffolds compared to natural TIM barrels. We developed a multistep design workflow combining physics- and AI-based tools including Triad and RFdiffusion to scaffold a Kemp eliminase activity into a de novo TIM barrel. 1 out of 5 designs showed an activity comparable to previous first design rounds of Kemp eliminases. This workflow provides a generalizable approach to the design of tailor-made active sites through scaffolding.

POSTER NO 29: Karolina Krzesińska

MAVISp: A Modular Structure-Based Framework for Protein Variant Effects

Karolina Krzesińska^1,2, Matteo Arnaudi^1,2, Joachim Breitenstein^1,2, Simone Scrima², Giaccomo Miletti³,  Apolinar Maya-Mendoza³, Matteo Lambrughi², Matteo Tiberti², Elena Papaleo¹,

¹Cancer Systems Biology, Section for Bioinformatics, Department of Health and Technology, Technical University of Denmark, 2800, Lyngby, Denmark. ²Cancer Structural Biology, Danish Cancer Institute, 2100, Copenhagen, Denmark. ³DNA Replication and Cancer, Danish Cancer Institute, 2100, Copenhagen, Denmark

Contact: kzokr@dtu.dk

The role of genomic variants in diseases, including cancer, continues to expand thanks to the advent of advanced sequencing techniques integrated into clinical practice [1]. The rapid growth in the identification of genomic variants has led to the classification of many variants as Variants of Uncertain Significance (VUS) or those with conflicting evidence, posing challenges in their interpretation and application. Additionally, current methods for predicting pathogenic variants do not necessarily provide information on the mechanisms underlying pathogenicity [2-4].

 

We introduce MAVISp (Multi-layered Assessment of VarIants by Structure for proteins), a modular structural framework for variant effects, to handle high-throughput saturation variant analysis with a standardised workflow, integrating results with various pathogenicity predictors [5]. Currently, MAVISp offers analyses for 400 different proteins, encompassing more than 3 million variants.

 

As proof of concept, we present a case study focusing on transcription factor and chromatin regulator ARID3A. Our analysis reveals which ARID3A mutations impact critical aspects of protein function, including structural stability, DNA and functional interactor-binding capacity, and alterations in distally located functional sites and pockets. Experimental validation of a predicted destabilising mutation within the DNA-binding domain of ARID3A corroborated the computational analysis, reinforcing the in silico predictions. Furthermore, validation of the interaction of ARID3A with TP53BP1 reveals a potential novel role in DNA damage response. Our framework aids in interpreting the effects of known and novel variants and holds great potential for advancing the understanding and application of mutational data in disease research.

POSTER NO 30: Kristine Degn

Unraveling Compensation in Cancer: A Novel Approach to Identifying Pathogenic and Rescue Mutations through a XGBoost Algorithm with p53 as a Model Protein

Kristine Degn(1), Matteo Tiberti(2), Julian Echave(3), Elena Papaleo(1,2)

(1) Cancer Systems Biology, Section for Bioinformatics, Department of Health and Technology, Technical University of Denmark, 2800 Lyngby, Denmark

(2) Cancer Structural Biology, Danish Cancer Institute, 2100 Copenhagen, Denmark

(3) - Instituto de Ciencias Físicas, Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina

Contact: krde@dtu.dk

Compensatory, or rescue, mutations occur at a distinct site from a pathogenic or deleterious mutation, offsetting its effects and exemplifying positive epistasis [1]. Identifying such rescue sites holds critical implications for understanding mechanisms of drug resistance in antibacterial, chemotherapeutic, and antiviral treatments. Thus, gaining a deeper understanding of compensatory effects might be utilized in future clinical strategies, showcasing the broader relevance of compensatory effects. Despite the significance of compensatory mutations, there is a notable gap in computational tools tailored to this field, relying primarily on anisotropic network models [2,3], which motivates our proposed analysis and potential approach. In examining the tumor suppressor protein p53 as a model, we identify features of known compensatory mutations and utilize these in an Extreme Gradient Boosting (XGBoost) algorithm, applied to p53 as a model, as a positive unlabeled problem, incorporating predicted evolutionary conservation, fitness and stability as features to enhance insights.

 

[1] Azbukina N, Zharikova A, Ramensky V. Intragenic compensation through the lens of deep mutational scanning. Biophys Rev 2022; 14:1161–1182

[2] Tiberti M, Pandini A, Fraternali F, et al. In silico identification of rescue sites by double force scanning. Bioinformatics 2018; 34:207–214

[3] Echave J. Fast computational mutation-response scanning of proteins. PeerJ 2021; 9:e11330

POSTER NO 31: Kristoffer Johansson

Global Analysis of Multi-Mutants to Improve Protein Function and Stability

Kristoffer E. Johansson, Kresten Lindorff-Larsen and Jakob R. Winther

Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen

Contact: kristoffer.johansson@bio.ku.dk

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. Multiplexed assays can screen thousands of protein variants in a single high-throughput experiment, for properties and at conditions that are relevant for the engineering task. Using such assays, we have demonstrated that large libraries of protein variants with several amino acid substitutions each are highly informative for engineering purposes. To realize this, we have developed a global multi-mutant analysis (GMMA) that can disentangle the effects of individual amino acid substitutions. This approach has proven robust and effective in three different studies: The stabilization of a designed proteins to >150 C, enhancement of green fluorescent protein, and stabilization of a molecular sensor across more functional constraints. In all cases, the optimization is achieved in a single round of experiments by introducing 5-9 mutations.

POSTER NO 32: Lars Santema

Discovery by cell-free protein synthesis and biochemical characterization of thermostable glycerol oxidases

Lars L. Santema[a], Laura Rotilio[b], Ruite Xiang[c], Gwen Tjallinks[a], Victor Guallar[c], Andrea Mattevi[b] & Marco W. Fraaije[a]

[a] Molecular Enzymology, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands. [b] Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy. [c] Electronic and atomic protein modelling group, Barcelona Supercomputing Center, E-08034 Barcelona, Spain.

Contact: l.l.santema@rug.nl

Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals[1]. By integrating in silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify enzymes that are active on glycerol. Three new thermostable alditol oxidases from Actinobacteria bacterium, Streptomyces thermoviolaceus and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent thermostabilities with melting temperatures higher than 75 ⁰C. Structural elucidation of the alditol oxidase from Actinobacteria bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen-bonds, a histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols.

 

References

1 Villa, A., Dimitratos, N., Chan-Thaw, C. E., Hammond, C., Prati, L., & Hutchings, G. J. (2015). Glycerol oxidation using gold-containing catalysts. Accounts of Chemical Research, 48(5), 1403–1412. https://doi.org/10.1021/ar500426g

 

Key words: Alditol oxidases, flavin, glycerol, cell-free expression, in silico bioprospecting, enzyme engineering.

 

*Corresponding author: Lars L. Santema; L.L.santema@rug.nl 31613809887

POSTER NO 33: Luis I. Gutierrez-Rus

Combining rational and computational design for de novo metalloenzymes

Luis I Gutierrez Rus, Sam Rogers, Aimee L Boyle & Derek N Woolfson

School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK / Max Planck-Bristol Centre for Minimal Biology, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UK,

Contact: luis.gutierrezrus@bristol.ac.uk

De novo design of  efficient artificial metalloenzymes aimed to catalyse novel enzymatic reactions of biotechnological interest constitutes a main challenge for protein design. Artificial metalloenzymes usually display interesting features that facilitate the catalysis of difficult reactions, but also provide chemical environments that enable chemo-, regio- and stereo-specificity. Coiled-coil based scaffolds such as three and four-helix bundles have been extensively used as starting points for metal binding design. In some cases, yielding artificial proteins with some catalytic properties. However, despite the abundant literature focused in the design of de novo metalloproteins, these studies are often focused in engineering or redesigning a specific structural region of the protein rather than systematically exploring and evaluating the whole scaffold. In this work, we present preliminary results regarding a design pipeline for artificial metalloenzymes that combine rational and computational design. Our goal is to create a general pipeline that can be applied to any protein scaffold and that allows us to systematically explore all plausible metal binding sites with specific constrains in order to find the best binding site for catalytic purposes. As a test case, we use a 4-helix bundle rationally designed by following defined rules for coiled-coil peptide assembly as a starting scaffold, and metal-catalysed hydrolysis of chemical bonds mediated by a water molecule acting as a Lewis acid as a target reaction. Our results show that our initial scaffold is easily functionalised to bind Zn2+ ions just by following a rational approach. However, our pipeline fails to predict tight binders by using cutting edge AI methodologies such us AlphaFold3 and Metal3D, highlighting important limitations in computational design of metalloproteins.

POSTER NO 34: Maria Correia (Davis)

Mechanism of consecutive multisite phosphorylation

1. C. Davis and M. Kjægaard

Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Contact: mcorr@mbg.au.dk

Multisite phosphorylation is an essential mechanism of cellular control, often used as a thresholding system to regulate a variety of essential physiological processes, including cell division. However, the fundamental mechanism of multisite phosphorylation is not well understood. In this work, I investigate whether introducing structured multivalency changes the reaction mechanism of the system to allow for the reaction to proceed in a processive manner. I use protein kinase A (PKA) and its cognate substrate in a multivalent disordered polypeptide with five equally spaced phosphorylatable sites to test if multivalency can lead to the rapid production of highly phosphorylated molecules in biomolecular condensates. Since phosphorylation is known to control phase separation and therefore the regulation of biomolecular condensates, the results of this work identify the fundamental principles of kinase-dependent condensate regulation, particularly with respect to the impact of multi-site phosphorylation on the regulatory dynamics controlling condensates. Condenstates and kinases form a mutually responsive system regulating many basic aspects of cellular physiology, therefore elucidating how they interact will provide greater insights into disease mechanisms and development of novel synthetic therapeutics.

POSTER NO 35: Matteo Cagiada

Predicting absolute protein folding stability using generative models

Matteo Cagiada

University of Copenhagen

Contact: matteo.cagiada@bio.ku.dk

While there has been substantial progress in our ability to predict changes in protein stability due to amino acid substitutions, progress has been slow in methods to predict the absolute stability of a protein. In outrwork, we showed how a generative model for protein sequence can be leveraged to predict absolute protein stability. We benchmarked our predictions across a broad set of proteins and find a mean error of 1.5~kcal/mol and a correlation coefficient of 0.7 for the absolute stability across a range of small--medium sized proteins up to ca. 150 amino acid residues. We analysed current limitations and future directions including how such model may be useful for predicting conformational free energies. Our approach is simple to use and freely available via an online implementation.

POSTER NO 36: Mette Errebo Rønne

Carbohydrate-binding module regulation of enzymatic transglycosylation

Mette Errebo Rønne¹, Bo Pilgaard², Marlene Vuillemin², Marie Sofie Møller¹

¹Applied Molecular Enzyme Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Denmark; ²Enzyme Discovery, Department of Biotechnology and Biomedicine, Technical University of Denmark

Contact: meero@dtu.dk

Oligosaccharides and their conjugates find wide applications in industry, serving as health promoting prebiotics and in production of therapeutics and cosmetics. They are produced by two main industrial methods: chemical synthesis from smaller compounds, which yield well-defined products and glycoconjugates, and the degradation of polysaccharides using chemical or enzymatic methods, resulting in complex mixtures. However, achieving a cost-effective, sustainable synthesis remains a significant challenge. Biotechnological approaches, particularly enzymatic methods, present eco-friendly alternatives to chemical synthesis and hold great potential for generating unique compounds.

Carbohydrate-active enzymes (CAZymes), notably glycoside hydrolases (GHs), can catalyze glycosidic bond formation through transglycosylation (TG). Optimizing the transglycosylation-to-hydrolysis (T/H) ratio requires careful engineering. While studies typically focus on the catalytic domain, emerging research suggests that carbohydrate-binding modules (CBMs) in multimodular GHs also influence TG activity, though the mechanisms remain poorly understood. This study aims to clarify the role of CBMs in TG reactions and to offer strategies and method development for enhancing TG yields. We hypothesize that CBMs promote TG by increasing local substrate concentration, product elongation, or active site conformation. Using enzymes from the multispecific GH5 family with confirmed TG activity on mannan or β-glucan found in subfamilies GH5_7, GH5_8, GH5_47 and GH5_55, we will investigate the effects of CBMs on TG and enzyme stability. Affinity and structural analyses will reveal how CBMs contribute to substrate binding and channeling. The gained insights from this work will be applied in GH fusion design for improved TG reactions and substrate specificity through CBM engineering, aiming to advance enzyme engineering for the industrial synthesis of oligosaccharides.

POSTER NO 37: Milena R. Lalic & Elisabeth Thomsen

Interdomain Interaction Through Structural Disorder in Nuclear Receptors

Elisabeth G. K. Thomsen^1,2,3, Milena Lalic^1,2,3, F Emil Thomasen^2,3, Andreas Prestel^1,2,3, Lukas W. Bauer^1,2, Johan G. Olsen^1,2,3, Cy Jeffries⁴, Kresten Lindorff-Larsen^2,3, Birthe B. Kragelund^1,2,3.

¹REPIN, ²Structural Biology and NMR Laboratory, and ³The Linderstr¯m-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark,⁴European Molecular Biology Laboratory (EMBL), Hamburg Unit, Deutsches Elektronen-Synchroton, Hamburg, Germany.

Contact: milena.lalic@bio.ku.dk

Structural disorder within nuclear receptors modulates intra- and intermolecular interactions, conferring potential allosteric communication channels essential for transcription. We combined SAXS, NMR spectroscopy, and molecular dynamics to address how the interdomain interaction in PPARγ and PPARɑ, the master regulator of adipogenesis and liver metabolism, respectively, is facilitated and orchestrated by structural disorder. Our results demonstrate that the long, disordered AB domain of PPARs interacts dynamically with the DBD as well as the LBD. Notably, upon DNA binding, the AB domain is released to a degree that depends on the PPAR isomer. This event exposes possible activation domains responsible for controlling activation of gene expression. This study enhances our understanding of how the disordered AB domain communicates with various domains of PPARs, contributing to a broader comprehension of the regulatory mechanisms within nuclear receptors.

POSTER NO 38: Nadja Joachim

Phosphorous recovery using optimised phosphate binding proteins

Nadja Joachim and Kaare Teilum

Structural Biology and NMR laboratory (SBiNlab), Department of Biology, University of Copenhagen, Denmark

Contact: nadja.joachim@bio.ku.dk

The major aim of this project is to construct a phosphate-specific chromatographic column that can recover inorganic phosphate (Pi) from wastewater, using the bacterial phosphate binding protein PstS. Phosphorous is essential for plant growth and modern agriculture relies on its use in fertilizers. However, phosphorous reserves are limited, and by the use in farming, it ends up in sewage or washed out of the soil and into our waterways, lakes and oceans mainly as inorganic phosphate, Pi.1  PstS is a highly selective and high affinity binder of Pi, and could therefore serve as a tool to recover phosphate.2 To make a reusable column, we require highly stable protein, and conditions of binding and eluting Pi. By screening a library of 96 diverse PstS sequences, around 30 variants with a higher thermal stability than E. coli PstS were identified. To engineer a pH sensitive switch to release bound phosphate, we make use of the mutation T141D in the binding site of E. coli PstS, which leads to preferred binding of the monobasic compared to dibasic phosphate, and therefore disfavours binding above pH 8.3 As the binding site is highly conserved, this mutation can also be introduced in the stable homologues identified in the screen, which resulted in a more stable PstS variant with pH sensitive affinity for Pi, demonstrated by ITC. We aim to further increase this sensitivity by introducing mutations in the hinge region, followed by optimising protein stability to make a durable column.

 

1 Cordell, D. and White, S. (2014) Life’s Bottleneck: Sustaining the World’s Phosphorus for a Food Secure Future. Annu. Rev. Env. Resour. 39, 161–188.

2 Luecke, H. and Quiocho, F. A. (1990) High specificity of a phosphate transport protein determined by hydrogen bonds. Nature 347, 402–406.

3 Wang, Z., Luecke, H., Yao, N. and Quiocho, F. A. (1997) A low energy short hydrogen bond in very high resolution structures of protein receptor-phosphate complexes. Nat. Struct. Biol.

POSTER NO 39: Nancy Forde

Repurposing non-motor proteins to make novel molecular motors

Chapin S. Korosec (1,2), Suzana Kovacic (1), Michael H.W. Kirkness (1), Ivan N. Unksov (3), Paul M. G. Curmi (4), Heiner Linke (3) & Nancy R. Forde (1)

(1) Simon Fraser University, Canada

(2) York University, Canada

(3) Lund University, Sweden

(4) University of New South Wales, Australia

Contact: nforde@sfu.ca

Nature uses proteins as its building block of choice for molecular motors: devices capable of transducing free energy into mechanical work. As a community, we have been studying these natural motors to learn the “rules” by which these incredible machines operate. But how well do we understand these mechanisms?  Our international team challenged ourselves to build novel molecular motors using building blocks of proteins that are not associated with naturally occurring motors. We have recently succeeded in characterizing one of these designs, the Lawnmower, which we believe provides the first demonstration of motility of a synthetic, protein-based motor. In this presentation, I’ll describe the design of the Lawnmower [1], the design of the peptide-based lawn that powers its motion [2], and the experiments and analysis that convinced us that its dynamics were powered by the designed “burnt-bridge ratchet” mechanism [3].

 

[1] S. Kovacic et al., IEEE Trans. NanoBiosci. 14, 305 (2015)

[2] M.W.H. Kirkness, C.S. Korosec and N.R. Forde, Langmuir 34, 13550 (2018)

[3] C.S. Korosec et al., Nature Communications 15, 1511 (2024)

POSTER NO 40: Nicolas Sebastian Gonzalez Foutel

Effect of protein condensates on kinase signaling

Nicolas S. Gonzalez-Foutel (1); Magnus Kjaergaard (1-3)

(1) Department of Molecular Biology and Genetics, Aarhus University

(2) The Danish Research Institute for Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine

(3) Center for Proteins in Memory - PROMEMO, Danish National Research Foundations

Contact: nfoutel@mbg.au.dk

Macromolecular condensates are formed by spontaneous and reversible self-interactions of proteins and nucleic acids i.e. scaffolds, followed by their phase separation inside the cell. These condensates can recruit enzymes and substrates, known as clients, boosting or quenching biochemical reactions and therefore acting as switches in cellular signaling pathways. Phosphorylation by protein kinases has been shown to occur inside these condensates, but little is known about their functional consequences and how this could affect the kinase signaling network. We hypothesize that condensates change phosphorylation rates and that a slow diffusion inside the condensates is the dominant effect modifying these reactions. To study this, we target both Protein kinase A and substrates of different quality (kcat/KM) and length, to synthetic intrinsically disordered protein condensates formed by repetitive octapeptides. We then measured phosphorylation kinetics by 32P radioactivity assays. Preliminary results show that condensates reduce phosphorylation rates irrespective of any substrate. This decrease is more pronounced for lengthier kinase substates regardless of their quality, which are recruited and kinetically trapped in a slow diffusion regime. However, given the same substrate quality, the reduction in phosphorylation rates is less noticeable for shorter substrates, where their diffusion rates across the condensates are faster. This work provides better understanding on how condensates affect kinase reactions and might help to elucidate generalizable effects of condensates.

POSTER NO 41: Nina Louise Jacobsen

Calmodulin regulates the signaling pathways of the Interleukin-4 Receptor

Nina L. Jacobsen (1,2,3), Pernille Seiffert (1,2), Daniel Sieme (1,2),  Flemming H. Larsen (3), Birthe B. Kragelund (1,2)

(1) REPIN and (2) Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Denmark, (3) LEO Pharma A/S, Ballerup, Denmark

Contact: nina.jacobsen@bio.ku.dk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 42: Oriana Sacco

Biochemical characterization of a novel archaeal 4-α-glucanotransferase from Pyrobaculum arsenaticum: Insights into disproportionation and cyclization activity

Oriana Sacco^1,2,5, Yu Wang⁵, Nicola Curci², Federica De Lise², Ali Shaikh Ibrahim^1,2, Mauro Di Fenza², Andrea Strazzulli^1,3,4, Marco Moracci^1,3,4, Birte Svensson⁵, and Beatrice Cobucci-Ponzano²

¹ Department of Biology, University of Naples ‘Federico II’ Naples, Italy. ² Institute of Biosciences and BioResources, National Research Council of Italy Naples, Italy. ³ Task Force on Microbiome Studies, University of Naples “Federico II”, 80138 Naples, Italy, ⁴ NBFC, National Biodiversity Future Center, Palermo 90133, Italy. ⁵ Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.

Contact: oriana.sacco@unina.it

Amylomaltases (AMs), classified in the carbohydrate-active enzyme database (CAZy) in glycoside hydrolase family 77 (GH77) (Drula et al., 2022), are carbohydrate-active enzymes with a monospecific 4-α-glucanotransferase activity (EC 2.4.1.25). AMs catalyse the cleavage of α-1,4 glucosidic bonds in α-1,4-D-glucans and the subsequent transfer of the resulting glucan chain to the O-4 position of an α-1,4-D-glucan acceptor. Their unique transferase activity has made AMs valuable for biotechnological applications, such as synthesizing sugar substitutes, producing cycloamyloses, and modifying starch for industrial purposes (Li et al., 2024; Leoni et al., 2021). Despite the annotation in CAZy of over 19,000 GH77 sequences, only a small number of AMs have been reported as characterised enzymes, of which very few from thermophilic sources. Obtaining new thermostable enzymes is critical in the field of biotechnology, as their intrinsic stability characteristics are well-suited to the demands of various industrial applications. Therefore, biochemical characterisation of newly identified heat-stable enzymes is required. In this study, we focus on the characterisation of a novel GH77 enzyme from the archaeon Pyrobaculum arsenaticum, identified in a metagenomic dataset derived from an 85°C, pH 5.5 mud/water hot spring in the solfataric field of Pisciarelli, Agnano (Naples, Italy) (Strazzulli et al., 2021). The enzyme exhibits optimal activity at 95°C and pH 5.5, with a specific activity of 1500 U/mg on maltotriose. It has also been demonstrated that the enzyme is able to perform disproportionation activity producing maltooligosaccharides with different degrees of polymerisation and it has been tested on amylose to evaluate its cyclisation activity.

 

References

Drula, E., Garron, M. L., Dogan, S., Lombard, V., Henrissat, B., & Terrapon, N. (2022). Nucleic Acids Research, 50(D1), D571–D577.

Leoni, C., Gattulli, B. A. R., Pesole, G., Ceci, L. R., & Volpicella, M. (2021). Biomolecules, 11(9), 1335.

Strazzulli, A., Cobucci-Ponzano, B., Iacono, R., Giglio, R., Maurelli, L., Curci, N., Schiano-di-Cola, C., Santangelo, A., Contursi, P., Lombard, V., Henrissat, B., Lauro, F. M., Fontes, C., & Moracci, M. (2020). The FEBS Journal, 287(6), 1116–1137.

Li, X., Wang, Y., Wu, J., Jin, Z., Dijkhuizen, L., Svensson, B., & Bai, Y. (2024). Designing starch derivatives with desired structures and functional properties via rearrangements of glycosidic linkages by starch-active transglycosylases. Critical Reviews in Food Science and Nutrition, 64(23), 8265–8278.

POSTER NO 43: Poul Thrane

A Michaelis-Menten model for heterogeneous enzyme kinetics on a rugged energy landscape

Poul Thrane

Department of Science and Environment, Roskilde University

Contact: poult@ruc.dk

Enzyme reactions in complex systems with multiple substrates are prevalent in both technical and industrial applications of enzymes. However, the consequences of substrate heterogeneity on enzyme kinetics are poorly understood. It is critical to develop both models and methods to capture this kinetic heterogeneity, as ensemble-averaged measurements may conceal kinetic distributions and obscure the impact of heterogeneity in enzyme-catalyzed reactions. Here we examine the impact of substrate heterogeneity on steady-state enzyme kinetics in systems with multiple substrates. We extend the classical Michaelis-Menten (MM) model and its energy representation to a system with an arbitrary number of substrates and show that the observable kinetics are characterized by a simple MM model. Furthermore, we find that the observable catalytic rate constant increases with the substrate heterogeneity, while the observable MM-constant decreases. These results were derived through mathematical analysis and verified by numerical simulations.

POSTER NO 44: Rasmus Krogh Norrild

Large-scale quantification of protein and RNA energetic contributions to biomolecular condensation

Rasmus K. Norrild, Sören von Bülow, Einar Halldórsson, Kresten Lindorff-Larsen, Joseph M. Rogers, and Alexander K. Buell

Technical University of Denmark

University of Copenhagen

Contact: rkrno@dtu.dk

Biomolecular condensates have emerged as a new concept to understand cellular membrane-less organelles involved in compartmentalisation, regulation, and signalling. Relying on a multitude of interactions, phase separation of dynamic and weakly interacting protein and RNA molecules contribute to assembly. These interactions are difficult to quantify by traditional methods that limit the thermodynamic data available for predictive models. Here we present mRNA-Display for Condensate Partitioning (mRNA-DCP) to directly measure energetic contributions of peptide and RNA molecules to formation of biomolecular condensates, at a large scale. Using the intrinsically disordered region (IDR) of Dead-box helicase 4 (DDX4N1), we measure the partitioning coefficients of almost 100,000 peptides and corresponding mRNA molecules between the protein rich and depleted phases. Using partitioning as a proxy for intrinsic propensity for phase separation, we show that DDX4N1 peptide fragments provide high-resolution data on regions of the protein domain responsible for homotypic condensate formation. In addition, peptide tiles from all other experimentally verified IDRs form a catalogue of potential clients of the condensates and sequence features governing partitioning can be generalised to explain phase separation of unrelated IDRs. We simultaneously measured partitioning of the same number of synthetic mRNA molecules which showed longer, purine-rich, and less structured RNA partition stronger. The unprecedented scale of the data generated by this method allows quantitative evaluation of how condensation behaviour and potential specificity are encoded in protein and RNA.

POSTER NO 45: Samuel Peña Díaz

Alternative protein-based strategies for plastic degradation

Samuel Peña-Díaz, Pedro Ferreira, Andreas Møllebjerg, Malthe K. Bendtsen, Alexander Sandahl, Maria João Ramos and Daniel E. Otzen

Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark;

Faculdade de Ciencias, Universidad do Porto, Porto, Portugal;

Danish Technological Institute, Aarhus, Denmark,

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Contact: samuelpd@inano.au.dk

Plastic pollution has become a major environmental problem, significantly exacerbated by the lack of viable recycling technologies for many plastic types. The use of biological approaches, namely enzymes, stands out as a great green and sustainable alternative for selective plastic degradation and recycling. However, the number of plastic degrading enzymes is still scarce. In a multidisciplinary collaboration, our group has developed different strategies to efficiently depolymerize different types of plastic. The ENCORE strategy focuses on the optimization of a fluorescence-based protocol to screen nylon degrading capacity in bacteria from environmental samples. This strategy has yielded different bacteria with novel nylon degrading capacity and has identified potential enzymes involved in this process (NYLONases), currently under scrutiny and characterization. Nevertheless, enzymatic plastic degradation is intrinsically challenged by a narrow catalytic cavity and low stability under harsh conditions. To address this, we have embarked on the use of amyloid fibrils as catalytic scaffolds for plastic degradation, exploiting their high stability and repetitive structures. We have combined experimental analysis of the inner fibrils’ catalytic properties with computational tools to transplant the catalytic site of a PETase onto the fibrillar structure and optimized a screening protocol to validate constructs’ activity. We aim to further improve this activity through the use of recently developed AI tools for protein design.

POSTER NO 46: Simina Cuciurean

Transient helicity in the intrinsically disordered protein ACTR measured by hydrogen exchange

Simina Cuciurean, Christian Parsbæk Buch, and Kaare Teilum

University of Copenhagen

Contact: simina.cuciurean@bio.ku.dk

The 1424-amino acid nuclear receptor coactivator 3 (NCOA3), a key transcriptional activator interacting with steroid hormone receptors, is implicated in various cancers. Understanding its regulatory structure is crucial, yet the full-length NCOA3 poses challenges for conventional techniques due to its length and disordered nature. The assessment of labile amide protons exchange in proteins serves as a valuable tool for evaluating the local stability of folded globular proteins. In the case of intrinsically disordered proteins (IDPs) such as the activation domain of the nuclear receptor coactivator 3 ACTR, the hydrogen exchange rate is significantly faster compared to folded proteins and the current hydrogen exchange reference data lack the quality needed to characterize NCOA3's transient structures. This study shows that in variants of ACTR, which exhibit differential population in α-helix structures, it is possible to determine the stability of the helix by hydrogen exchange NMR, if one of the variants is used as an internal reference. Our findings demonstrate the efficacy of HX in discerning transient structures within IDPs and emphasize the critical necessity for more precise reference data specific to unstructured proteins.

POSTER NO 47: Soumik Ray

Divergent effects of pathological alpha-synuclein mutations on phase separation

Soumik Ray1, Cecilia Chiodaroli, Sophie Hertel, Katharina Helga Schott, Antonin Kunka, Azad Farzadfard, Kristina Mojtic, Céline Galvagnion Büll, and Alexander Kai Büll

Contact: souray@dtu.dk

THE ABSTRACT IS NOT PUBLIC.

POSTER NO 48: Steffie Elkjær

How do intrinsically disordered transcription factors compete for the same hub?

Steffie Elkjær, Charlotte O’Shea, and Karen Skriver

University of Copenhagen, department of Biology

Contact: steffie.elkjaer@bio.ku.dk

The three gene-regulating transcription factors, ANAC046, DREB2A, and ANAC013, are part of stress-induced plant systems regulated by the Radical-Induced Cell Death1 (RCD1). Interactions between the transcription factors and the folded hub domain RST in RCD1 are driven by a common RST-binding sequence motif located in the intrinsically disordered regions of the transcription factors. These binding regions all undergo coupled folding and binding to form a common minimal structural core consisting of a short strand followed by a turn. Here we performed stopped-flow kinetic analysis of the core structure regions upon interactions with RST as a platform for mechanistic understanding. This showed that the core structure of the transcription factors exhibits different affinities, primarily determined by the dissociation rate constants and are unable to displace each other via a ternary complex. Changes in NMR chemical shift perturbations for RST upon titration with the three transcription factors showed similar patterns of affected residues at the binding pocket of RST. However, the affected area on RST expanded differently from the binding pocket with increasing affinities. Extending the core structure to include flanking regions affected both affinities and competition. Interactions of RST with DREB2A and NAC046 are stabilized by the flanking regions, while the interaction with NAC013 is weakened. The presence of the flanking regions enables ‘active’ displacement, where one transcription factor most likely pirates the binding site on RST from another transcription factor before unperturbed dissociation. Overall, the study provides a mechanistic understanding of the RST-hub interactome and demonstrates how flanking regions can contribute to highly complex competition at interaction hotspots.

POSTER NO 49: Stephanie Heusser

Understanding Desensitization Mechanisms in Acid-Sensing Ion Channels Through Protein Modifications

Stephanie A. Heusser¹, Debayan Sarkar¹, Iacopo Galleano^1,2, Caroline Marcher Holm¹, Asli B. Topaktas^1,3, Johs Dannesboe^1,4, Christian B. Borg¹, Stephan A. Pless¹.

¹Dep. of Drug Design and Pharmacology, University of Copenhagen, Denmark, ²Dep. of Cell and Chemical Biology, Leiden University Medical Centre, Netherlands. ³Dep. of Cellular and Molecular Medicine, University of Copenhagen, Denmark, ⁴Dep. of Biomedical Sciences, University of Copenhagen, Denmark

Contact: stephanie.heusser@sund.ku.dk

Acid-sensing ion channels 1a (ASIC1a) are proton-gated sodium channels that activate below pH 6.8 and contribute to fast synaptic transmission, learning, and memory. Apart from activation, ASIC1a also undergoes several types of desensitization suggested to prevent acidosis-mediated neurotoxicity. Stabilizing desensitization could thus be of relevance for targeting the channel during ischemic stroke, chronic pain and neurological disorders.

We use chemical biology approaches and fluorometry to elucidate the dynamic structure-function relationships underlying ASIC desensitization. We specifically studied the role of an H-bonding network involving a conserved lysine (Lys 211) in the extracellular domain using split intein-mediated protein semi-synthesis to incorporate non-canonical Lys analogs. We saw that changes to the side chain length, geometry, or charge enhanced acute desensitization likely via a discrupted coordination of a chloride ion which in turn destabilizes the open state of the channel.

We further investigated a mutation in the β11- β12 linker, previously reported to significantly reduce desensitization. Voltage-clamp fluorometry experiments with fluorescently labeled channel domains revealed that, although the extracellular domain of the mutant channels still undergoes pH-dependent conformational changes, these changes appear to be disconnected from the functional state of the pore. Similar decoupled states were also observed even long after exposure of ASIC1a to the tarantula toxin PcTx1, which introduces desensitization at non-activating pH levels. Notably, this decoupled state has possible pharmacological implications, as it limits the binding of a known neuropeptide.

Together, these findings provide deeper insights into desensitization and underscore its complexity by showcasing several distinct molecular mechanisms involved in ASIC1a desensitization.

POSTER NO 50: Sören von Bülow

Prediction of phase separation propensities of disordered proteins from sequence

Sören von Bülow, Giulio Tesei, Kresten Lindorff-Larsen

Structural Biology and NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Contact: soren.bulow@bio.ku.dk

Phase separation (PS) is one possible mechanism governing the selective cellular enrichment of biomolecular constituents for processes such as transcriptional activation, mRNA regulation, and immune signalling. PS is mediated by multivalent interactions of biological macromolecules including intrinsically disordered proteins and regions (IDRs). Despite considerable advances in experiments, theory and simulations, the prediction of the thermodynamics of IDR phase behaviour remains challenging.

We combined coarse-grained molecular dynamics simulations and active learning to develop a fast and accurate machine learning model to predict the free energy and saturation concentration for PS directly from sequence [1]. We validate the model using both experimental and computational data. We apply our model to all IDRs in the human proteome and find that ~5% of these are predicted to undergo homotypic PS. We use our model to understand the relationship between single-chain compaction [2] and PS, and find that changes from charge- to hydrophobicity-mediated interactions can break the symmetry between intra- and inter-molecular interactions.

Our work refines the established rules governing the relationships between sequence features and PS propensities, and our prediction models will be useful for interpreting and designing cellular experiments on the role of PS, and for the design of IDRs with specific PS propensities.

 

References

[1] S. von Bülow, G. Tesei, and K. Lindorff-Larsen. Prediction of phase separation propensities of disordered proteins from sequence. bioRxiv 2024. 10.1101/2024.06.03.597109

[2] G. Tesei, A.I. Trolle, et al. Conformational ensembles of the human intrinsically disordered proteome. Nature 2024. 10.1038/s41586-023-07004-5

POSTER NO 51: Thea Klarsø Schulze

Predicting residue-level free energies of hydrogen exchange from structure

Thea K. Schulze and Kresten Lindorff-Larsen

University of Copenhagen

Contact: thea.schulze@bio.ku.dk

TBA

POSTER NO 52: Vili Lampinen

Targeted Design of Protein Binders to Probe Receptor Signaling in Neuronal Connectivity and Memory

Vili Lampinen, Magnus Kjærgaard

Aarhus University

Contact: vili.lampinen@mbg.au.dk

Neurotrophin signaling plays a crucial role in neuronal connectivity and memory formation. This project aims to develop genetically encodable miniprotein binders to study and manipulate neuroreceptors, specifically TrkB and p75NTR. Utilizing advanced AI tools such as RFdiffusion and proteinMPNN, we will design high-affinity binders targeting the extracellular domain of TrkB and both the extra- and intracellular domains of p75NTR. After confirming specific, high-affinity binding, we will use the binders in cell culture assays to manipulate the neuroreceptors towards cell phenotypes associated with activation or blocking of these receptors. By homo- and heterodimerizing successful binders together, we aim to force neuroreceptor monomers in each other’s proximity on the cell surface, which is known to induce signaling and affect substrate specificity in the case of the TrkB and p75NTR pair. The ultimate goal is to provide new tools for dissecting the molecular mechanisms underlying learning and memory, especially in elucidating the activation mechanisms of p75NTR, which are still unclear to a large extent. Future potential applications include developing therapeutic strategies for neurodegenerative diseases such as Alzheimer’s, with the immediate gain from the project being molecular tools for researching neuroreceptor functions and a robust pipeline for designing binders against similar targets.

POSTER NO 53: Yu Wang

Comparative Study of Three Starch Branching Enzymes Identifies Effective Enzyme Tools for Granular Starch Modification

Yu Tian^a, Saadia Riaz^a, Edita Jurak^b, Marc J.E.C. van der Maarel^b, Birte Svensson^a, Marie Sofie Møller^{c, *}, Yu Wang^{a, *}

^a Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark. ^b Bioproduct Engineering, Engineering and Technology Institute Groningen, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. ^c Applied Molecular Enzyme Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark

Contact: yuawa@dtu.dk

Glycogen branching enzyme (GBE) is widely applied to modify starch. However, only GBEs from Geobacillus thermoglucosidans (GtGBE) and Rhodothermus obamensis (RoGBE) have been used on starch granules. To further develop this modification, granules of waxy, normal, and three types of high-amylose maize starch are treated with GBEs from Petrotoga mobilis (PmGBE), Rhodothermus marinus (RmGBE) and benchmarked by RoGBE. PmBE fastest and most effectively added short branches to starch granules and reduced crystallinity and degree of surface order it had lowest activity. Furthermore, granules treated by PmGBE increased most in resistant starch (RS) content by Englyst digestibility analysis. PmGBE in accordance with its high activity showed superior capacity in Langmuir binding to granules. It is proposed that the N-terminal domain of unknown function (DUF) only present in PmGBE and GBE surface binding sites influence activity and substrate specificity. PmGBE showed strong potential for producing highly surface branched starch granules with enhanced resistance to digestive enzyme.