FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.

Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose-binding protein, a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing that Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method that also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ∼1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Oxygen Isotopologues Resolved from Water Oxidation Electrocatalysis by Electron Paramagnetic Resonance Spectroscopy.

Electrocatalytic water oxidation is a key transformation in many strategies designed to harness solar energy and store it as chemical fuels. Understanding the mechanism(s) of the best electrocatalysts for water oxidation has been a fundamental chemical challenge for decades. Here, we quantitate evolved dioxygen isotopologue composition via gas-phase EPR spectroscopy to elucidate the mechanisms of water oxidation on metal oxide electrocatalysts with high precision. Isotope fractionation is paired with computational and kinetic modeling, showing that this technique is sensitive enough to differentiate O-O bond-forming steps. Strong agreement between experiment and theory indicates that for the nickel-iron layered double hydroxide─one of the best earth-abundant electrocatalysts to be studied─water oxidation proceeds via a dioxo coupling mechanism to form a side-bound peroxide rather than a hydroxide attack to form an end-bound peroxide.

chiLife: An open-source Python package for in silico spin labeling and integrative protein modeling.

Here we introduce chiLife, a Python package for site-directed spin label (SDSL) modeling for electron paramagnetic resonance (EPR) spectroscopy, in particular double electron-electron resonance (DEER). It is based on in silico attachment of rotamer ensemble representations of spin labels to protein structures. chiLife enables the development of custom protein analysis and modeling pipelines using SDSL EPR experimental data. It allows the user to add custom spin labels, scoring functions and spin label modeling methods. chiLife is designed with integration into third-party software in mind, to take advantage of the diverse and rapidly expanding set of molecular modeling tools available with a Python interface. This article describes the main design principles of chiLife and presents a series of examples.

Modeling of Cu(II)-based protein spin labels using rotamer libraries.

The bifunctional spin label double-histidine copper-(II) capped with nitrilotriacetate [dHis-Cu(II)-NTA], used in conjunction with electron paramagnetic resonance (EPR) methods can provide high-resolution distance data for investigating protein structure and backbone conformational diversity. Quantitative utilization of this data is limited due to a lack of rapid and accurate dHis-Cu(II)-NTA modeling methods that can be used to translate experimental data into modeling restraints. Here, we develop two dHis-Cu(II)-NTA rotamer libraries using a set of recently published molecular dynamics simulations and a semi-empirical meta-dynamics-based conformational ensemble sampling tool for use with the recently developed chiLife bifunctional spin label modeling method. The accuracy of both the libraries and the modeling method are tested by comparing model predictions to experimentally determined distance distributions. We show that this method is accurate with absolute deviation between the predicted and experimental modes between 0.0-1.2 Å with an average of 0.6 Å over the test data used. In doing so, we also validate the generality of the chiLife bifunctional label modeling method. Taken together, the increased structural resolution and modeling accuracy of dHis-Cu(II)-NTA over other spin labels promise improvements in the accuracy and resolution of protein models by EPR.

Bayesian Probabilistic Inference of Nonparametric Distance Distributions in DEER Spectroscopy.

Double electron-electron resonance (DEER) spectroscopy measures distance distributions between spin labels in proteins, yielding important structural and energetic information about conformational landscapes. Analysis of an experimental DEER signal in terms of a distance distribution is a nontrivial task due to the ill-posed nature of the underlying mathematical inversion problem. This work introduces a Bayesian probabilistic inference approach to analyze DEER data, assuming a nonparametric distance distribution with a Tikhonov smoothness prior. The method uses Markov Chain Monte Carlo sampling with a compositional Gibbs sampler to determine a posterior probability distribution over the entire parameter space, including the distance distribution, given an experimental data set. This posterior contains all of the information available from the data, including a full quantification of the uncertainty about the model parameters. The corresponding uncertainty about the distance distribution is visually captured via an ensemble of posterior predictive distributions. Several examples are presented to illustrate the method. Compared with bootstrapping, it performs faster and provides slightly larger uncertainty intervals.

Juvenile downstream migration patterns of an anadromous fish, allis shad (Alosa alosa), before and after the population collapse in the Gironde system, France.

Diadromous fish have exhibited a dramatic decline since the end of the 20th century. The allis shad (Alosa alosa) population in the Gironde-Garonne-Dordogne (GGD) system, once considered as a reference in Europe, remains low despite a fishing ban in 2008. One hypothesis to explain this decline is that the downstream migration and growth dynamics of young stages have changed due to environmental modifications in the rivers and estuary. We retrospectively analysed juvenile growth and migration patterns using otoliths from adults caught in the GGD system 30 years apart during their spawning migration, in 1987 and 2016. We coupled otolith daily growth increments and laser ablation inductively-coupled plasma mass spectrometry measurements of Sr:Ca, Ba:Ca, and Mn:Ca ratios along the longest growth axis from hatching to an age of 100 days (i.e., during the juvenile stage). A back-calculation allowed us to estimate the size of juveniles at the entrance into the brackish estuary. Based on the geochemistry data, we distinguished four different zones that juveniles encountered during their downstream migration: freshwater, fluvial estuary, brackish estuary, and lower estuary. We identified three migration patterns during the first 100 days of their life: (a) Individuals that reached the lower estuary zone, (b) individuals that reached the brackish estuary zone, and (c) individuals that reached the fluvial estuary zone. On average, juveniles from the 1987 subsample stayed slightly longer in freshwater than juveniles from the 2016 subsample. In addition, juveniles from the 2016 subsample entered the brackish estuary at a smaller size. This result suggests that juveniles from the 2016 subsample might have encountered more difficult conditions during their downstream migration, which we attribute to a longer exposure to the turbid maximum zone. This assumption is supported by the microchemical analyses of the otoliths, which suggests based on wider Mn:Ca peaks that juveniles in 2010s experienced a longer period of physiological stress during their downstream migration than juveniles in 1980s. Finally, juveniles from the 2016 subsample took longer than 100 days to exit the lower estuary than we would have expected from previous studies. Adding a new marker (i.e., Ba:Ca) helped us refine the interpretation of the downstream migration for each individual.

Thermally Ultrarobust S = 1/2 Tetrazolinyl Radicals: Synthesis, Electronic Structure, Magnetism, and Nanoneedle Assemblies on Silicon Surface.

Open-shell organic molecules, including S = 1/2 radicals, may provide enhanced properties for several emerging technologies; however, relatively few synthesized to date possess robust thermal stability and processability. We report the synthesis of S = 1/2 biphenylene-fused tetrazolinyl radicals 1 and 2. Both radicals possess near-perfect planar structures based on their X-ray structures and density-functional theory (DFT) computations. Radical 1 possesses outstanding thermal stability as indicated by the onset of decomposition at 269 °C, based on thermogravimetric analysis (TGA) data. Both radicals possess very low oxidation potentials <0 V (vs. SCE) and their electrochemical energy gaps, Ecell ≈ 0.9 eV, are rather low. Magnetic properties of polycrystalline 1 are characterized by superconducting quantum interference device (SQUID) magnetometry revealing a one-dimensional S = 1/2 antiferromagnetic Heisenberg chain with exchange coupling constant J'/k ≈ -22.0 K. Radical 1 in toluene glass possesses a long electron spin coherence time, Tm ≈ 7 µs in the 40-80 K temperature range, a property advantageous for potential applications as a molecular spin qubit. Radical 1 is evaporated under ultrahigh vacuum (UHV) forming assemblies of intact radicals on a silicon substrate, as confirmed by high-resolution X-ray photoelectron spectroscopy (XPS). Scanning electron microscope (SEM) images indicate that the radical molecules form nanoneedles on the substrate. The nanoneedles are stable for at least 64 hours under air as monitored by using X-ray photoelectron spectroscopy. Electron paramagnetic resonance (EPR) studies of the thicker assemblies, prepared by UHV evaporation, indicate radical decay according to first-order kinetics with a long half-life of 50 ± 4 days at ambient conditions.

Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

Site-directed spin-labeling (SDSL)─in combination with double electron-electron resonance (DEER) spectroscopy─has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides─including a gem-diethyl-substituted nitroxide with enhanced stability in cells─with rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy.

Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.

Comparative evaluation of spin-label modeling methods for protein structural studies.

Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a powerful technique for the investigation of protein structure and dynamics. Accurate spin-label modeling methods are essential to make full quantitative use of site-directed spin-labeling electron paramagnetic resonance data for protein modeling and model validation. Using a set of double electron-electron resonance data from seven different site pairs on maltodextrin/maltose-binding protein under two different conditions using five different spin labels, we compare the ability of two widely used spin-label modeling methods, based on accessible volume sampling and rotamer libraries, to predict experimental distance distributions. We present a spin-label modeling approach inspired by canonical side-chain modeling methods and compare modeling accuracy with the established methods.

Dipolar pathways in dipolar EPR spectroscopy.

Dipolar electron paramagnetic resonance (EPR) experiments such as double electron-electron resonance (DEER) measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and other macromolecular systems. To determine these distributions from the experimental signal, it is critical to employ an accurate model of the signal. For dilute samples of doubly spin-labeled molecules, the signal is a product of an intramolecular and an intermolecular contribution. We present a general model based on dipolar pathways valid for dipolar EPR experiments with spin-1/2 labels. Our results show that the intramolecular contribution consists of a sum and the intermolecular contribution consists of a product over individual dipolar pathway contributions. We examine several commonly used dipolar EPR experiments in terms of dipolar pathways and show experimental results confirming the theoretical predictions. This multi-pathway model makes it possible to analyze a wide range of dipolar EPR experiments within a single theoretical framework.

Dipolar pathways in multi-spin and multi-dimensional dipolar EPR spectroscopy.

Dipolar electron paramagnetic resonance (EPR) experiments, such as double electron-electron resonance (DEER), measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and other macromolecular systems. We present an extension to our previously published general model based on dipolar pathways valid for multi-dimensional dipolar EPR experiments with more than two spin-1/2 labels. We examine the 4-pulse DEER and TRIER experiments in terms of dipolar pathways and show experimental results confirming the theoretical predictions. This extension to the dipolar pathways model allows the analysis of previously challenging datasets and the extraction of multivariate distance distributions.

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.

Spectroscopic Investigation of a Metal-Metal-Bonded Fe6 Single-Molecule Magnet with an Isolated S = 19/2 Giant-Spin Ground State.

The metal-metal-bonded molecule [Bu4N][(HL)2Fe6(dmf)2] (Fe6) was previously shown to possess a thermally isolated spin S = 19/2 ground state and found to exhibit slow magnetization relaxation below a blocking temperature of ∼5 K [J. Am. Chem. Soc. 2015, 137, 13949-13956]. Here, we present a comprehensive spectroscopic investigation of this unique single-molecule magnet (SMM), combining ultrawideband field-swept high-field electron paramagnetic resonance (EPR) with frequency-domain Fourier-transform terahertz EPR to accurately quantify the spin Hamiltonian parameters of Fe6. Of particular importance is the near absence of a 4th-order axial zero-field splitting term, which is known to arise because of quantum mechanical mixing of spin states on account of the relatively weak spin-spin (superexchange) interactions in traditional polynuclear SMMs such as the celebrated Mn12-acetate. The combined high-resolution measurements on both powder samples and an oriented single crystal provide a quantitative measure of the isolated nature of the spin ground state in the Fe6 molecule, as well as additional microscopic insights into factors that govern the quantum tunneling of its magnetization. This work suggests strategies for improving the performance of polynuclear SMMs featuring direct metal-metal bonds and strong ferromagnetic spin-spin (exchange) interactions.

Determining electron-nucleus distances and Fermi contact couplings from ENDOR spectra.

The hyperfine coupling between an electron spin and a nuclear spin depends on the Fermi contact coupling aiso and, through dipolar coupling, the distance r between the electron and the nucleus. It is measured with electron-nuclear double resonance (ENDOR) spectroscopy and provides insight into the electronic and spatial structure of paramagnetic centers. The analysis and interpretation of ENDOR spectra is commonly done by ordinary least-squares fitting. As this is an ill-posed, inverse mathematical problem, this is challenging, in particular for spectra that show features from several nuclei or where the hyperfine coupling parameters are distributed. We introduce a novel Tikhonov-type regularization approach that analyzes an experimental ENDOR spectrum in terms of a complete non-parametric distribution over r and aiso. The approach uses a penalty function similar to the cross entropy between the fitted distribution and a Bayesian prior distribution that is derived from density functional theory calculations. Additionally, we show that smoothness regularization, commonly used for a similar purpose in double electron-electron resonance (DEER) spectroscopy, is not suited for ENDOR. We demonstrate that the novel approach is able to identify and quantitate ligand protons with electron-nucleus distances between 4 and 9 Å in a series of vanadyl porphyrin compounds.

Bayesian Probabilistic Analysis of DEER Spectroscopy Data Using Parametric Distance Distribution Models.

Double electron-electron resonance (DEER) spectroscopy measures distance distributions between spin labels in proteins, yielding important structural and energetic information about conformational landscapes. Analysis of an experimental DEER signal in terms of a distance distribution is a nontrivial task due to the ill-posed nature of the underlying mathematical inversion problem. This work introduces a Bayesian probabilistic inference approach to analyze DEER data, using a multi-Gauss mixture model for the distance distribution. The method uses Markov chain Monte Carlo (MCMC) sampling to determine a posterior probability distribution over model parameter space. This distribution contains all the information available from the data, including a full quantification of the uncertainty about the parameters. The corresponding uncertainty about the distance distribution is captured via an ensemble of posterior predictive distributions. Several synthetic examples illustrate the method. An experimental example shows the importance of model checking and comparison using residual analysis and Bayes factors. Overall, the Bayesian approach allows for more robust inference about protein conformations from DEER spectroscopy.

Modeling of motional EPR spectra using hindered Brownian rotational diffusion and the stochastic Liouville equation.

Electron paramagnetic resonance (EPR) spectra of molecular spin centers undergoing reorientational motion are commonly simulated using the stochastic Liouville equation (SLE) with a rigid-body hindered Brownian diffusion model. Current SLE theory applies to specific spin systems such as nitroxides and to high-symmetry orientational potentials. In this work, we extend the SLE theory to arbitrary spin systems with any number of spins and any type of spin Hamiltonian interaction term, as well as to arbitrarily complex orientational potentials. We also examine the limited accuracy of the frequency-to-field conversion used to obtain field-swept EPR spectra and present a more accurate approach. The extensions allow for the simulation of EPR spectra of all types of spin labels (nitroxides, copper2+, and gadolinium3+) attached to proteins in low-symmetry environments.

How Metal Ion Lewis Acidity and Steric Properties Influence the Barrier to Dioxygen Binding, Peroxo O-O Bond Cleavage, and Reactivity.

Herein we quantitatively investigate how metal ion Lewis acidity and steric properties influence the kinetics and thermodynamics of dioxygen binding versus release from structurally analogous Mn-O2 complexes, as well as the barrier to Mn peroxo O-O bond cleavage, and the reactivity of Mn oxo intermediates. Previously we demonstrated that the steric and electronic properties of MnIII-OOR complexes containing N-heterocyclic (NAr) ligand scaffolds can have a dramatic influence on alkylperoxo O-O bond lengths and the barrier to alkylperoxo O-O bond cleavage. Herein, we examine the dioxygen reactivity of a new MnII complex containing a more electron-rich, less sterically demanding NAr ligand scaffold, and compare it with previously reported MnII complexes. Dioxygen binding is shown to be reversible with complexes containing the more electron-rich metal ions. The kinetic barrier to O2 binding and peroxo O-O bond cleavage is shown to correlate with redox potentials, as well as the steric properties of the supporting NAr ligands. The reaction landscape for the dioxygen chemistry of the more electron-rich complexes is shown to be relatively flat. A total of four intermediates, including a superoxo and peroxo species, are observed with the most electron-rich complex. Two new intermediates are shown to form following the peroxo, which are capable of cleaving strong X-H bonds. In the absence of a sacrificial H atom donor, solvent, or ligand, serves as a source of H atoms. With TEMPOH as sacrificial H atom donor, a deuterium isotope effect is observed (kH/kD = 3.5), implicating a hydrogen atom transfer (HAT) mechanism. With 1,4-cyclohexadiene, 0.5 equiv of benzene is produced prior to the formation of an EPR detected MnIIIMnIV bimetallic species, and 0.5 equiv after its formation.