Ensemble structure of the N-terminal domain (1-267) of FUS in a biomolecular condensate.

Solutions of some proteins phase separate into a condensed state of high protein concentration and a dispersed state of low concentration. Such behavior is observed in living cells for a number of RNA-binding proteins that feature intrinsically disordered domains. It is relevant for cell function via the formation of membraneless organelles and transcriptional condensates. On a basic level, the process can be studied in vitro on protein domains that are necessary and sufficient for liquid-liquid phase separation (LLPS). We have performed distance distribution measurements by electron paramagnetic resonance for 13 sections in an N-terminal domain (NTD) construct of the protein fused in sarcoma (FUS), consisting of the QGSY-rich domain and the RGG1 domain, in the denatured, dispersed, and condensed state. Using 10 distance distribution restraints for ensemble modeling and three such restraints for model validation, we have found that FUS NTD behaves as a random-coil polymer under good-solvent conditions in both the dispersed and condensed state. Conformation distribution in the biomolecular condensate is virtually indistinguishable from the one in an unrestrained ensemble, with the latter one being based on only residue-specific Ramachandran angle distributions. Over its whole length, FUS NTD is slightly more compact in the condensed than in the dispersed state, which is in line with the theory for random coils in good solvent proposed by de Gennes, Daoud, and Jannink. The estimated concentration in the condensate exceeds the overlap concentration resulting from this theory. The QGSY-rich domain is slightly more extended, slightly more hydrated, and has slightly higher propensity for LLPS than the RGG1 domain. Our results support previous suggestions that LLPS of FUS is driven by multiple transient nonspecific hydrogen bonding and π-sp2 interactions.

Integrative ensemble modeling of proteins and their complexes with distance distribution restraints.

Many proteins and protein complexes exhibit regions that are intrinsically disordered. Whereas an arsenal of techniques exists to characterize structured proteins or protein regions, characterization of the vast conformational space occupied by intrinsically disordered regions remains a challenging task due the ensemble-averaging nature of many techniques that provide mean value restraints. More representative information can be gained in the form of distribution restraints, such as EPR-derived distance distributions. Previously we developed the ensemble modeling tool MMM, where we partition the macromolecule into structured and unstructured domains and utilize an integrative structural approach with a focus on EPR-derived distance restraints. Here we present the successor program of MMM: MMMx. All the modeling functionality was ported to MMMx and is now accessed by a uniform script format, allowing to combine the different modules at will to modeling pipelines. During the conception of MMMx many of the tools were improved or updated. We discuss the general functionality of MMMx and its modules, and illustrate some of the modeling tools by application examples.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Structural biology of RNA-binding proteins in the context of phase separation: What NMR and EPR can bring?

Liquid-liquid phase separation of RNA-binding proteins underlies the formation of membraneless organelles, whose composition is dynamic and whose existence may be transient. These organelles are involved in regulation of RNA processing and translation and, if they behave abnormally, in pathologies. Because disorder phenomena are essential in their formation and dynamics, established methodology is insufficient for characterizing their structure. In this review, we consider the current and potential contribution of NMR and EPR spectroscopy to the understanding of structure and dynamics of phase-separating RNA-binding proteins in, both, their dispersed and condensed state in vitro. We discuss which experiments are applicable under what conditions and which information can be obtained from them. Because for these phenomena, the accessible information depends crucially on metastable phase equilibria, we also consider aspects of sample preparation for NMR and EPR experiments.

Characterization of Weak Protein Domain Structure by Spin-Label Distance Distributions.

Function of intrinsically disordered proteins may depend on deviation of their conformational ensemble from that of a random coil. Such deviation may be hard to characterize and quantify, if it is weak. We explored the potential of distance distributions between spin labels, as they can be measured by electron paramagnetic resonance techniques, for aiding such characterization. On the example of the intrinsically disordered N-terminal domain 1-267 of fused in sarcoma (FUS) we examined what such distance distributions can and cannot reveal on the random-coil reference state. On the example of the glycine-rich domain 188-320 of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) we studied whether deviation from a random-coil ensemble can be robustly detected with 19 distance distribution restraints. We discuss limitations imposed by ill-posedness of the conversion of primary data to distance distributions and propose overlap of distance distributions as a fit criterion that can tackle this problem. For testing consistency and size sufficiency of the restraint set, we propose jack-knife resampling. At current desktop computers, our approach is expected to be viable for domains up to 150 residues and for between 10 and 50 distance distribution restraints.

NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation.

Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.

Open and Closed Radicals: Local Geometry around Unpaired Electrons Governs Magic-Angle Spinning Dynamic Nuclear Polarization Performance.

The development of magic-angle spinning dynamic nuclear polarization (MAS DNP) has allowed atomic-level characterization of materials for which conventional solid-state NMR is impractical due to the lack of sensitivity. The rapid progress of MAS DNP has been largely enabled through the understanding of rational design concepts for more efficient polarizing agents (PAs). Here, we identify a new design principle which has so far been overlooked. We find that the local geometry around the unpaired electron can change the DNP enhancement by an order of magnitude for two otherwise identical conformers. We present a set of 13 new stable mono- and dinitroxide PAs for MAS DNP NMR where this principle is demonstrated. The radicals are divided into two groups of isomers, named open (O-) and closed (C-), based on the ring conformations in the vicinity of the N-O bond. In all cases, the open conformers exhibit dramatically improved DNP performance as compared to the closed counterparts. In particular, a new urea-based biradical named HydrOPol and a mononitroxide O-MbPyTol yield enhancements of 330 ± 60 and 119 ± 25, respectively, at 9.4 T and 100 K, which are the highest enhancements reported so far in the aqueous solvents used here. We find that while the conformational changes do not significantly affect electron spin-spin distances, they do affect the distribution of the exchange couplings in these biradicals. Electron spin echo envelope modulation (ESEEM) experiments suggest that the improved performance of the open conformers is correlated with higher solvent accessibility.