Frans A. A. Mulder

Intrinsically disordered proteins (IDPs) play essential roles in cellular signaling and regulation, often relying on transient structural elements to mediate interactions. NMR chemical shifts are widely used to detect secondary structures in IDPs, but complementary methods are needed to validate and refine these measurements. Hydrogen exchange is a powerful probe of local structure and dynamics in folded proteins, yet its accuracy for detecting small differences in transient helicity in IDPs remains understudied. Here, we systematically evaluate hydrogen exchange measured by NMR and MS (HDX-MS) in four variants of the activator for thyroid hormone and retinoid receptors (ACTR) activation domain that differ in helical propensity. Using NMR-based exchange rates, we introduce pseudo-protection factors referenced to the wild-type protein, enabling a robust comparison among variants without relying on "intrinsic" peptide-based chemical exchange rates. These pseudo-protection factors correlate strongly with helicity derived from chemical shifts, demonstrating that hydrogen exchange can resolve subtle structural differences in highly dynamic regions and vice versa. Our findings establish hydrogen exchange as a sensitive and reproducible method for characterizing transient structure in IDPs, complementing NMR chemical shift analysis.

Disordered proteins play essential roles in myriad cellular processes, yet their structural characterization remains a major challenge due to their dynamic and heterogeneous nature. Here we present a community-driven initiative to address this problem by advocating a unified framework for determining conformational ensembles of disordered proteins. Our aim is to integrate state-of-the-art experimental techniques with advanced computational methods, including knowledge-based sampling, enhanced molecular dynamics and machine learning models. The modular framework comprises three interconnected components: experimental data acquisition, computational ensemble generation and validation. The systematic development of this framework will ensure the accurate and reproducible determination of conformational ensembles of disordered proteins. We highlight the open challenges necessary to achieve this goal, including force-field accuracy, efficient sampling, and environmental dependence, advocating for collaborative benchmarking and standardized protocols.

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) RIα 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 RIα 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 RIα by nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations. The intrinsically disordered regions of RIα cause broadening of NMR signals throughout the protein indicative of widespread dynamic interactions. Mutation of arginine residues in the RIα inhibitory sequence leads to a reduction in signal broadening accompanied by loss of phase separation. Independently, binding of RIα 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 RIα with the intrinsically disordered linker, and the intermolecular interactions in the condensate mirror those found internally in RIα dimers. Our results support the view that for PKA RIα, homotypic phase separation is underpinned by intermolecular interactions that are similar to intramolecular interactions.

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. It is shown here that the partitioning of components in a complex mixture can be robustly obtained from a single set of 2D nuclear magnetic resonance (NMR) spectra recorded on the dilute and dense phases separately using “time‐zero extrapolated” heteronuclear single quantum coherence (HSQC₀) spectroscopy. 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 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 membrane‐less organelles.

Drebrin (developmentally regulated brain protein) is a vital component of the Postsynaptic Density (PSD). It performs important biological roles as it interacts with the postsynaptic protein Homer and anchors the complete protein network to the cellular skeleton through actin filaments. Drebrin contains unique structural elements including an evolutionarily unconventional actin-depolymerizing factor homology (ADFH) domain that has lost its strong actin-binding ability, and a Single Alpha-Helix (SAH) motif harbored by long flexible regions. In vivo studies have suggested that a disordered segment in Drebrin plays a key role in binding filamentous actin, yet the atomic-level characterization of the binding interface between these proteins has not been reported. To bridge this gap, we designed the intrinsically disordered construct D233 and employed 3D (HN)CO(CO)NH NMR spectroscopy to accomplish a near-complete backbone resonance assignment. This work serves as an essential step toward a detailed structural and functional investigation of the interaction between Drebrin and F-Actin.

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 HSQC ₀ . 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 ¹ H NMR, it is validated that the HSQC ₀ 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.

Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease‐related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so‐called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step‐wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild‐type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR‐based Dark‐state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.

MCherry is one of the most successfully applied monomeric red fluorescent proteins (RFPs) for in vivo and in vitro imaging. However, questions pertaining to the photostability of the RFPs remain and rational further engineering of their photostability requires information about the fluorescence quenching mechanism in solution. To this end, NMR spectroscopic investigations might be helpful, and we present the near-complete backbone NMR chemical shift assignment to aid in this pursuit.

Oxidative stress and formation of cytotoxic oligomers by the natively unfolded protein α-synuclein (α-syn) are both connected to the development of Parkinson's disease. This effect has been linked to lipid peroxidation and membrane disruption, but the specific mechanisms behind these phenomena remain unclear. To address this, we have prepared α-syn oligomers (αSOs) in vitro in the presence of the lipid peroxidation product 4-oxo-2-nonenal and investigated their interaction with live cells using in-cell NMR as well as stimulated emission depletion (STED) super-resolution and confocal microscopy. We find that the αSOs interact strongly with organellar components, leading to strong immobilization of the protein's otherwise flexible C-terminus. STED microscopy reveals that the oligomers localize to small circular structures inside the cell, while confocal microscopy shows mitochondrial fragmentation and association with both late endosome and retromer complex before the SOs interact with mitochondria. Our study provides direct evidence for close contact between cytotoxic α-syn aggregates and membraneous compartments in the cell.