Biology subjects

Kjaergaard, M.

Publications and source records attributed to Kjaergaard, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Affinity and distance dependence of SLiM-mediated dephosphorylation by protein phosphatase 1

Protein phosphatases counterbalance kinases by dephosphorylating phospho-proteins to regulate signaling pathways. But unlike kinases, their shallow catalytic groove has limited selectivity for the phospho-peptide motifs. Phosphatases such as protein phosphatase-1 (PP1) recognize their substrates through short linear motifs (SLiMs) within intrinsically disordered regions, yet the mechanism of SLiM-mediated recruitment remains unclear. We developed an intrinsically disordered phospho-protein substrate to enable quantitative modelling of tethered PP1 catalysis by varying SLiM affinity and distance. We show that a PP1-binding SLiM linked to a phospho-peptide by a flexible spacer is sufficient to enhance dephosphorylation. Dephosphorylation occurs most efficiently at a spacing of 20-30 residues, in agreement with predictions from theoretical polymer models. Low micromolar dissociation constants are most efficient for dephosphorylation, which can be modelled numerically as a trade-off between substrate binding and substrate inhibition. Bivalent SLiMs enhance or decrease catalysis depending on the combined affinity. Together, these results define a quantitative and generalizable framework for how linear motifs direct dephosphorylation by PP1.

biophysics↗

bioRxiv2026

Global analysis of thermal and chemical denaturation using CheMelt: Thermodynamic dissection of highly thermostable de novo designed proteins

De novo protein design often produces thermostable proteins that denature above 100 {degrees}C, which complicates the analysis of their stability. Thermostable proteins can be unfolded by combined chemical and thermal denaturation followed by global analysis of multiple melting curves. Here, we have developed CheMelt, a new online tool for global analysis of unfolding data via an intuitive graphical user interface. We use nanoscale differential scanning fluorimetry followed by CheMelt data analysis to dissect the combined thermal and chemical denaturation of thirty-five de novo designed protein binders. Thirteen present sufficient fluorescence changes to extract thermodynamic parameters of unfolding. These de novo designed proteins have systematically lower {Delta}Cp and m-values than comparable natural proteins. We show that a high thermostability of a designed protein does not necessarily imply a high equilibrium stability, and demonstrate the potential of CheMelt in dissecting thermodynamic properties for protein design and engineering. CheMelt can be accessed at https://spc.embl-hamburg.de/app/chemelt

biophysics↗

bioRxiv2026

Sequence determinants of the hypomobility of intrinsically disordered proteins in SDS-PAGE

Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.

biophysics↗

bioRxiv2026

Slow diffusion limits phosphorylation in a biomolecular condensate

Biomolecular condensates form dynamic compartments that regulate biochemical reactions in cells. Condensates recruit many kinases and regulate their enzymatic activity. Condensates alter the rate of enzymatic reactions through several opposing effects, so it is unclear whether these mostly enhance or retard phosphorylation. Here, we use a synthetic condensate formed by intrinsically disordered proteins to show that slow diffusion in the condensate controls phosphorylation kinetics in the dense phase. We vary the length of substrates by appending phase-separating repeat proteins of different lengths, in order to study how phosphorylation depends on partitioning, diffusion and volume fraction across substrate motifs with different intrinsic kinetics. The condensate environment is generally inhibitory to phosphorylation, although the enzyme remains intact. This inhibition is partially offset by an enhanced reaction rate in the dilute phase, likely due to soluble nanoclusters. Phosphorylation rates are strongly correlated to diffusion coefficients of substrates in the condensate, suggesting mass-transport limitation. Our results suggest that condensates can modify the substrate usage of a kinase via different trade-offs between diffusion and partitioning. We suggest that diffusion limitations are likely a common feature of many macromolecular reactions in condensates, and that high fluidity is crucial for condensates to act as reaction crucibles.

biophysics↗

bioRxiv2026

Long-term single-molecule Ca2+ flux recordings reveal mode-switching regulation of Ca2+-ATPases

Calcium (Ca{superscript 2}) is a universal second messenger that governs processes ranging from muscle contraction and secretion to gene expression and cell fate. Ca{superscript 2}-ATPases establish and maintain steep Ca{superscript 2} gradients across intracellular membranes, yet how regulatory inputs modulate the underlying single-pump Ca{superscript 2} currents has remained inaccessible. Here we develop a non-saturating, self-regenerating single-vesicle assay that monitors over hours the zeptoampere (10-{superscript 2}{superscript 1} A) currents produced by individual Ca{superscript 2}-ATPases. In parallel, we establish a workflow to record single-molecule currents from human sarco/endoplasmic reticulum Ca{superscript 2}-ATPases (hSERCA) in native endoplasmic reticulum vesicles. Using reconstituted LMCA1, a bacterial SERCA homologue, we observe stochastic switching between minute-long pumping and inactive modes, as well as uncoupled Ca{superscript 2} leakage events that are suppressed by vanadate. Extravesicular pH controls a previously unrecognized dormant pre-activation mode that delays the onset of pumping, without measurably altering pumping rates or active-mode lifetimes. Extending the assay to endogenous hSERCA reveals delayed activation and ultraslow pumping/inactive mode-switching without detectable transprotein Ca{superscript 2} leakage. ATP and Ca{superscript 2} regulate the probability of hSERCA activation by modulating dormant-mode occupancy. Together, these results extend ultraslow mode-switching, previously observed only for proton pumps, to Ca{superscript 2}-ATPases and identify probability-gated entry into productive cycling as a distinct regulatory axis of human Ca{superscript 2}-ATPase regulation that can modulate the timing and heterogeneity of Ca{superscript 2} store refilling without changing on-cycle kinetics.

biophysics↗

bioRxiv2025

Phase separation of protein kinase A regulatory subunits is driven by similar inter- and intra-molecular interactions involving the inhibitory segment

Phase separation of biological macromolecules has emerged as a widespread mechanism for cellular organization, governing the spatiotemporal regulation of biochemical processes including signaling pathways. The regulatory subunit of the cAMP-dependent protein kinase A (PKA) RI phase separates following cAMP-induced kinase activation, thereby restricting and enhancing PKA activity in space and time. The N-terminal dimerization/docking (D/D) domain and the linker region of RI have been identified as necessary and sufficient for phase separation, but their interactions at atomic resolution have remained elusive. We report here a structural investigation of the minimal phase-separating region of RI by nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations. The intrinsically disordered regions of RI cause broadening of NMR signals throughout the protein indicative of widespread dynamic interactions. Mutation of arginine residues in the RI inhibitory sequence leads to a reduction in signal broadening accompanied by loss of phase separation. Independently, binding of RI to an A-kinase anchoring protein (AKAP) leads to spectral changes that are indicative of a loss of interactions between the D/D domain and the linker region, suggestive of a competition between intramolecular interactions and partner binding. Corroborating the experimental observations, multiscale MD simulations detect pervasive interactions of the folded D/D domain of RI with the intrinsically disordered linker, and the intermolecular interactions in the condensate mirror those found internally in RI dimers. Our results support the view that for PKA RI, homotypic phase separation is underpinned by intermolecular interactions that are similar to intramolecular interactions.

biophysics↗

bioRxiv2025

Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis

Computational protein design can create binders against targets of interest, but identifying binders with sufficient affinity still requires biochemical screening of many designs. In this work, we test flow-induced dispersion analysis (FIDA) as a method for screening binders in a time and cost-effective manner. FIDA uses Taylor dispersion analysis to determine the hydrodynamic radius of fluorescently labelled biomolecules and their complexes. Here, we use FIDA to assess the binding of RFdiffusion-designed protein binders against the small helical peptide ALFA-tag and the GK domain of PSD-95. Successful binders can be identified in a single measurement using heat-treated bacterial lysates allowing rapid identification of binders with high affinity, and thermostability. Subsequent titration experiments show micromolar affinities for ALFA-tag binders and nanomolar affinities for GK domain binders. The lack of immobilization, the minimal sample volume, and the compatibility with complex biological samples, positions FIDA as a valuable tool for the screening and characterization of computationally generated protein binders.

biophysics↗

bioRxiv2025

Quantification of small molecule partitioning in a biomolecular condensate with 2D NMR spectroscopy

Several intrinsically disordered proteins have been shown to undergo phase separation into a dense and dilute phase and this process is intimately linked with the regulation of cellular processes. It is therefore highly relevant to know how metabolites partition between these phases. We show here that the partitioning of components in a complex mixture can be robustly obtained from a single set of 2D NMR spectra recorded on the dilute and dense phases separately using a so-called "time zero extrapolated approach" known as HSQC0. The spectral separation power of 2D NMR spectroscopy circumvents the need for physical isolation or workup of the mixture components in the two samples. Using quantitative 1D 1H NMR, it is validated that the HSQC0 approach effectively removes all the undermining effects that plague quantification in common 2D NMR experiments, including differential attenuation due to relaxation in the two phases, pulse imperfections, partial decoupling, off-resonance effects, and incomplete coherence transfer in the case of scalar coupling variation. These results should be of widespread interest as partitioning into biomolecular condensates is crucial for the calibration of computational physicochemical models of phase separation and key to the further understanding of cellular biochemistry involving membraneless organelles.

biophysics↗

bioRxiv2025

Reactive oxygen generation by minimal copper binding peptide motifs

Intrinsically disordered proteins such as amyloid-{beta} (A{beta}), prion protein (PRP) and -synuclein bind copper-ions through histidine-rich motifs. Several of these copperbinding motifs catalyze formation of reactive oxygen species (ROS), which is believed to be involved in these proteins role in neurodegenerative disorders. The catalytic mechanism rely on binding in an in-between state, which is energetically available to both Cu(I) and Cu(II) and thus allow redox cycling. Presently, our knowledge of ROS generation is based on a few peptides studied for their pathological role and has thus not explored the minimal requirements for this function. Here, we investigate copper coordination and ROS generation in a series of synthetic histidine-rich peptides. We found that a minimal motif of two histidine residues joined by a few glycine residues is sufficient to generate ROS at two and a half times the rate of the corresponding A{beta} complex. Copper-coordination and ROS generation are flexible with regard to the spacing of these two histidine residues suggesting that physiologically relevant Cubinding motifs are degenerate. Spectroscopic studies indicate that ROS activity correlate to coordination by two imidazole nitrogen atoms in a dynamic complex. This study suggests that copper-coordination and ROS production might be more prevalent in intrinsically disordered proteins than realized.

biophysics↗

bioRxiv2024

Oxygen partitioning into biomolecular condensates is governed by excluded volume

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 intrinsically disordered repeat proteins with systematic sequence variations using microelectrodes and phosphorescence lifetime imaging microscopy (PLIM). Unlike other hydrophobic metabolites, oxygen is partially excluded from the condensate with partitioning constants more strongly modulated by changes in protein length than hydrophobicity. For repeat proteins, the dense phase protein concentration drops with chain length resulting in a looser condensate. We found that oxygen partitioning is anti-correlated with dense phase protein concentration. Several mechanisms could explain such an anti-correlation including excluded volume or salting out effects. 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 nanometer length-scales, which may tune the oxygen concentration available for biochemical reactions within the cell.

biophysics↗

bioRxiv2024

Structure of a Ca2+ bound phosphoenzyme intermediate in the inward-to-outward transition of Ca2+-ATPase 1 from Listeria monocytogenes

Active transport by Ca2+-ATPases of the P-type ATPase family maintain a very low cytosolic calcium concentration and steep electrochemical gradients. Detailed mechanisms of this transport have been described from structures of mammalian sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) stabilized by inhibitors at specific intermediate steps of the transport cycle. An essentially irreversible step is crucial to prevent reflux in active transport against steep gradients. Single-molecule FRET (smFRET) study of the bacterial Ca2+-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states, suggesting that calcium release from this intermediate is the irreversible step. Here, we present a 3.5[A] cryo-EM structure for a four-glycine insertion mutant (G4-LMCA1) in a lipid nanodisc obtained under turnover conditions and adopting such a calcium-bound intermediate, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain is similar to calcium-bound E1P-ADP like conformation of SERCA. Missing density for the E292 residue at the calcium site (equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism for the inward-to-outward transition in Ca2+-ATPases, where ADP release and re-organisation of the cytoplasmic domains precede calcium release.

molecular biology↗

bioRxiv2022

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Insulin formulations are the hallmark of interventions for treatment of diabetes. Understanding the mechanism that governs insulin self assembly or disassembly --and the role of stabilizing additives--are essential for improving insulin formulations. We report here the real-time direct observation of single insulin self-assembly and disassembly events using single molecule fluorescence microscopy. Our direct observations revealed previously unaccounted monomeric additions to occur to all types of assemblies and allowed us to quantify the existence, abundance and kinetic characterization of diverse assembly pathways involving monomeric dimers or tetrameric insulin species. We proposed and experimentally validated a model where the insulin self-assembly pathway is rerouted favoring monomeric or oligomeric assembly events by solution concentration, additives and formulations. Our rate simulation predicted the abundance of each oligomeric species across a concentration range of 6 orders of magnitude. Besides providing fundamental new insights, the results and toolbox here can be universally applied contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for other proteins.

biophysics↗

bioRxiv2022

The optimal docking strength for reversibly tethered kinases

Many kinases use reversible docking interactions to augment the specificity of their catalytic domains. Such docking interactions are often structurally independent of the catalytic domain, which allow for flexible combination of modules in evolution and in bioengineering. The affinity of docking interactions spans several orders of magnitude. This led us to ask how the affinity of the docking interaction affects enzymatic activity, and how to pick the optimal interaction module to complement a given substrate. Here, we develop equations that predict the optimal binding strength of a kinase docking interaction and validate it using numerical simulations and steady-state phosphorylation kinetics for tethered protein kinase A. We show that a kinase-substrate pair has an optimum docking strength that depends on their enzymatic constants, the tether architecture, the substrate concentration and the kinetics of the docking interactions. We show that a reversible tether enhances phosphorylation rates most when: I) The docking strength is intermediate, II) the substrate is non-optimal, III) the substrate concentration is low, IV) the docking interaction has rapid exchange kinetics, and V) the tether optimizes the effective concentration of the intra-molecular reaction. This work serves as a framework for interpreting mutations in kinase docking interactions and as a design guide for engineering enzyme scaffolds.

biophysics↗

bioRxiv2021

Estimation of effective concentrations enforced by complex linker architectures using conformational ensembles.

Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of the tether, which defines an effective concentration of the interactor. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments show that the effective concentration is mainly determined by the combination of the total length of flexible segments and the distance between the termini of the folded domains. We show that a folded domain in a disordered linker can increase the effective concentration beyond what can be achieved by a fully disordered linker by focusing the end-to-end distance at the appropriate spacing. This suggests that complex linker architecture may have advantages over simple flexible linkers and emphasizes that annotation as a linker should depend on the molecular context.

biophysics↗

bioRxiv2020

A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease

Existing tests for detecting liver fibrosis, inflammation and steatosis, three stages of liver disease that are still reversible are severely hampered by limited accuracy or invasive nature. Here, we present a paired liver-plasma proteomics approach to infer molecular pathophysiology and to identify biomarkers in a cross-sectional alcohol-related liver disease cohort of nearly 600 individuals. Metabolic functions were downregulated whereas fibrosis-associated signaling and novel immune responses were upregulated, but only half of tissue proteome changes were transmitted to the circulation. Machine learning models based on our biomarker panels outperformed existing tests, laying the foundation for a generic proteomic liver health assessment.

biochemistry↗

bioRxiv2020

Histidine-rich Ca2+-binding protein stimulates the transport cycle of SERCA through a conformation-dependent fuzzy complex.

The histidine-rich Ca2+-binding protein (HRC) stimulates the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) to increase Ca2+-uptake into the lumen. HRC also binds the triadin scaffold in a Ca2+-dependent manner, and HRC tunes both the uptake and release of Ca2+ depending on the concentration in the intracellular Ca2+-stores. We investigated how HRC stimulates SERCA pumping using biochemical and biophysical assays, and show that HRC is an intrinsically disordered protein that binds directly to SERCA via electrostatic interactions. The affinity of the interaction depends on the conformation of SERCA, and HRC binds most tightly in the calcium-released E2P state. This state marks the end of the rate-limiting [Ca2]E1P to E2P transition of SERCA, and suggests that HRC stimulates SERCA by preferentially stabilizing the end point of this transition. HRC remains disordered in the bound state and thus binds in a dynamic, fuzzy complex. The binding of HRC to SERCA shows that fuzzy complexes formed by disordered proteins may be conformation-specific, and use this specificity to modulate the functional cycle of complex molecular machines such as a P-type ATPase.

biochemistry↗

bioRxiv2020

The crystal structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a pump primed for dephosphorylation

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Here we present three crystal structures of Ca2+-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF3- mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca2+ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca2+-binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca2+ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters.

biochemistry↗

bioRxiv2020

Intrinsically disordered linkers control tethered kinases via effective concentration

Kinase specificity is crucial to the fidelity of signalling pathways, yet many pathways use the same kinases to achieve widely different effects. Specificity arises in part from the enzymatic domain, but also from the physical tethering of kinases to their substrates. Such tethering can occur via protein interaction domains in the kinase or via anchoring and scaffolding proteins, and can drastically increase the kinetics of phosphorylation. However, we do not know how such intra-complex reactions depend on the link between enzyme and substrate. Here we show that the kinetics of tethered kinases follow a Michaelis-Menten like dependence on effective concentration. We find that phosphorylation kinetics scale with the length of the intrinsically disordered linkers that join the enzyme and substrate, but that the scaling differs between substrates. Steady-state kinetics can only partially predict rates of tethered reactions as product release may obscure the rate of phospho-transfer. Our results suggest that changes in signalling complex architecture not only enhance the rates of phosphorylation reactions, but may also alter the relative substrate usage. This suggests a mechanism for how scaffolding proteins can allosterically modify the output from a signalling pathway.

biophysics↗