Pulsed Dipolar Electron Paramagnetic Resonance Research Articles

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid–liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock.

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.

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.

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Distance Measurements at Low Nanomolar Concentrations: The CuII-Trityl Case.

Recent sensitivity enhancements in pulse dipolar electron paramagnetic resonance spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin-labeling using CuII ions and trityl yields a >3-fold increase in sensitivity compared to that of the established CuII-nitroxide labeling strategy. Application of the recently developed variable-time relaxation-induced dipolar modulation enhancement (RIDME) method yields a further ∼2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, >2 times lower than the previously reported concentration. We expect that experiments at single-digit nanomolar concentrations are imminent, which have the potential to transform biological PDS applications.

Dissecting the Interaction between Cryptochrome and Timeless Reveals Underpinnings of Light-Dependent Recognition.

Circadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of Drosophila melanogaster, the clock is set by the light-induced degradation of the core oscillator protein timeless (TIM) by the principal light-sensor cryptochrome (CRY). The cryo-EM structure of CRY bound to TIM revealed that within the extensive CRY:TIM interface, the TIM N-terminus binds into the CRY FAD pocket, in which FAD and the associated phosphate-binding loop (PBL) undergo substantial rearrangement. The TIM N-terminus involved in CRY binding varies in isoforms that facilitate the adaptation of flies to different light environments. Herein, we demonstrate, through peptide binding assays and pulsed-dipolar electron spin resonance (ESR) spectroscopy, that the TIM N-terminal peptide alone exhibits light-dependent binding to CRY and that the affinity of the interaction depends on the initiating methionine residue. Extensions to the TIM N-terminus that mimic less light-sensitive variants have substantially reduced interactions with CRY. Substitutions of CRY residues that couple to the flavin rearrangement in the CRY:TIM complex have dramatic effects on CRY light activation. CRY residues Arg237 on α8, Asn253, and Gln254 on the PBL are critical for the release of the CRY autoinhibitory C-terminal tail (CTT) and subsequent TIM binding. These key light-responsive elements of CRY are well conserved throughout Type I cryptochromes of invertebrates but not by cryptochromes of chordates and plants, which likely utilize a distinct light-activation mechanism.

Lateral gating mechanism and plasticity of the β-barrel assembly machinery complex in micelles and Escherichia coli.

The β-barrel assembly machinery (BAM) mediates the folding and insertion of the majority of outer membrane proteins (OMPs) in gram-negative bacteria. BAM is a penta-heterooligomeric complex consisting of the central β-barrel BamA and four interacting lipoproteins BamB, C, D, and E. The conformational switching of BamA between inward-open (IO) and lateral-open (LO) conformations is required for substrate recognition and folding. However, the mechanism for the lateral gating or how the structural details observed in vitro correspond with the cellular environment remains elusive. In this study, we addressed these questions by characterizing the conformational heterogeneity of BamAB, BamACDE, and BamABCDE complexes in detergent micelles and/or Escherichia coli using pulsed dipolar electron spin resonance spectroscopy (PDS). We show that the binding of BamB does not induce any visible changes in BamA, and the BamAB complex exists in the IO conformation. The BamCDE complex induces an IO to LO transition through a coordinated movement along the BamA barrel. However, the extracellular loop 6 (L6) is unaffected by the presence of lipoproteins and exhibits large segmental dynamics extending to the exit pore. PDS experiments with the BamABCDE complex in intact E. coli confirmed the dynamic behavior of both the lateral gate and the L6 in the native environment. Our results demonstrate that the BamCDE complex plays a key role in the function by regulating lateral gating in BamA.

Confidence limits in pulse dipolar EPR spectroscopy: estimates for individual measurements.

The distribution of inter-label distances obtained by electron paramagnetic resonance (EPR) pulse dipolar spectroscopies (PDS), such as DEER aka PELDOR, gives a valuable characterization of structure on the nanometer scale. The impact of random experimental noise on such experiments is examined for three independent methods for analysing PDS data: the model-free method with Tikhonov regularization, model-free with Mellin-transformation, and a model-based method. All three methods show negative bias for the mean distance and positive bias for the distribution width. Both biases grow with increasing noise levels. The estimated confidence bands and the uncertainties obtained from a single experimental measurement by the standard bootstrapping or χ2-surface scanning approaches are inconsistent and can exclude the true distance distribution. Yet, both approaches can provide quite valuable support for hypothesis testing in PDS studies.

Spectroscopically Orthogonal Labelling to Disentangle Site-Specific Nitroxide Label Distributions

Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic metal ions and other organic radicals gaining popularity as alternative spin centres. Spectroscopically orthogonal spin labelling using different types of labels potentially increases the information content available from a single sample. When analysing experimental distance distributions between two nitroxide spin labels, the site-specific rotamer information has been projected into the distance and is not readily available, and the contributions of individual labelling sites to the width of the distance distribution are not obvious from the PDS data. Here, we exploit the exquisite precision of labelling double-histidine (dHis) motifs with CuII chelate complexes. The contribution of this label to the distance distribution widths in model protein GB1 has been shown to be negligible. By combining a dHis CuII labelling site with cysteine-specific nitroxide labelling, we gather insights on the label rotamers at two distinct sites, comparing their contributions to distance distributions based on different in silico modelling approaches and structural models. From this study, it seems advisable to consider discrepancies between different in silico modelling approaches when selecting labelling sites for PDS studies.

Pulse EPR spectroscopy and molecular modeling reveal the origins of the local heterogeneity of dietary fibers

Optimizing human diet by including dietary fibers would be more efficient when the fibers' chain interactions with other molecules are understood in depth. Thereby, it is important to develop methods for characterizing the fiber chain to be able to monitor its structural alterations upon intermolecular interactions. Here, we demonstrate the utility of the electron paramagnetic resonance (EPR) spectroscopy, complemented by simulations in probing the atomistic details of the chain conformations for spin-labeled fibers. Barley β-glucan, a native polysaccharide with linear chain, was utilized as a test fiber system to demonstrate the technique's capabilities. Pulse dipolar EPR data show good agreement with results of the fiber chain modeling, revealing sinuous chain conformations and providing polymer shape descriptors: the gyration tensor, spin-spin distance distribution function, and information about proton density near the spin probe. Results from EPR measurements point to the fiber aggregation in aqueous solution, which agrees with the results of the dynamic light scattering. We propose that the combination of pulse EPR measurements with modeling can be a perfect experimental tool for in-depth structural investigation of dietary fibers and their interaction under such conditions, and that the presented methodology can be extended to other weakly ordered or disordered macromolecules.

The effect of the zero-field splitting in light-induced pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy.

Laser-induced magnetic dipole (LaserIMD) spectroscopy and light-induced double electron-electron resonance (LiDEER) spectroscopy are important techniques in the emerging field of light-induced pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy (light-induced PDS). These techniques use the photoexcitation of a chromophore to the triplet state and measure its dipolar coupling to a neighboring electron spin, which allows the determination of distance restraints. To date, LaserIMD and LiDEER have been analyzed with software tools that were developed for a pair of two spins and that neglected the zero-field splitting (ZFS) of the excited triplet. Here, we explore the limits of this assumption and show that the ZFS can have a significant effect on the shape of the dipolar trace. For a detailed understanding of the effect of the ZFS, a theoretical description for LaserIMD and LiDEER is derived, taking into account the non-secular terms of the ZFS. Simulations based on this model show that the effect of the ZFS is not that pronounced in LiDEER for experimentally relevant conditions. However, the ZFS leads to an additional decay in the dipolar trace in LaserIMD. This decay is not that pronounced in Q-band but can be quite noticeable for lower magnetic field strengths in X-band. Experimentally recorded LiDEER and LaserIMD data confirm these findings. It is shown that ignoring the ZFS in the data analysis of LaserIMD traces can lead to errors in the obtained modulation depths and background decays. In X-band, it is additionally possible that the obtained distance distribution is plagued by long distance artifacts.

Interdomain Linkers Regulate Histidine Kinase Activity by Controlling Subunit Interactions.

Bacterial chemoreceptors regulate the cytosolic multidomain histidine kinase CheA through largely unknown mechanisms. Residue substitutions in the peptide linkers that connect the P4 kinase domain to the P3 dimerization and P5 regulatory domain affect CheA basal activity and activation. To understand the role that these linkers play in CheA activity, the P3-to-P4 linker (L3) and P4-to-P5 linker (L4) were extended and altered in variants of Thermotoga maritima (Tm) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allowed for a well-folded kinase domain that retained wild-type (WT)-like binding affinities for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registered ∼50-fold lower compared to WT. Neither a WT nor LV1 dimer containing a single P4 domain could autophosphorylate the P1 substrate domain. Autophosphorylation activity was rescued in variants with extended L3 and L4 linkers that favor helical structure and heptad spacing. Autophosphorylation depended on linker spacing and flexibility and not on sequence. Pulse-dipolar electron-spin resonance (ESR) measurements with spin-labeled adenosine 5'-triphosphate (ATP) analogues indicated that CheA autophosphorylation activity inversely correlated with the proximity of the P4 domains within the dimers of the variants. Despite their separation in primary sequence and space, the L3 and L4 linkers also influence the mobility of the P1 substrate domains. In all, interactions of the P4 domains, as modulated by the L3 and L4 linkers, affect domain dynamics and autophosphorylation of CheA, thereby providing potential mechanisms for receptors to regulate the kinase.

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Reveals Buffer-Modulated Cooperativity of Metal-Templated Protein Dimerization.

Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.

DEER Data Analysis Software: A Comparative Guide.

Pulsed dipolar electron paramagnetic resonance (PDEPR) spectroscopy experiments measure the dipolar coupling, and therefore nanometer-scale distances and distance distributions, between paramagnetic centers. Of the family of PDEPR experiments, the most commonly used pulsed sequence is four-pulse double electron resonance (DEER, also known as PELDOR). There are several ways to analyze DEER data to extract distance distributions, and this may appear overwhelming at first. This work compares and reviews six of the packages, and a brief getting started guide for each is provided.

A Low-Spin CoII/Nitroxide Complex for Distance Measurements at Q-Band Frequencies

Pulse dipolar electron paramagnetic resonance spectroscopy (PDS) is continuously furthering the understanding of chemical and biological assemblies through distance measurements in the nanometer range. New paramagnets and pulse sequences can provide structural insights not accessible through other techniques. In the pursuit of alternative spin centers for PDS, we synthesized a low-spin CoII complex bearing a nitroxide (NO) moiety, where both the CoII and NO have an electron spin S of 1/2. We measured CoII-NO distances with the well-established double electron–electron resonance (DEER aka PELDOR) experiment, as well as with the five- and six-pulse relaxation-induced dipolar modulation enhancement (RIDME) spectroscopies at Q-band frequencies (34 GHz). We first identified challenges related to the stability of the complex in solution via DEER and X-ray crystallography and showed that even in cases where complex disproportionation is unavoidable, CoII-NO PDS measurements are feasible and give good signal-to-noise (SNR) ratios. Specifically, DEER and five-pulse RIDME exhibited an SNR of ~100, and while the six-pulse RIDME exhibited compromised SNR, it helped us minimize unwanted signals from the RIDME traces. Last, we demonstrated RIDME at a 10 μM sample concentration. Our results demonstrate paramagnetic CoII to be a feasible spin center in medium magnetic fields with opportunities for PDS studies involving CoII ions.

Studying structure evolution of proteins using pulsed dipolar spectroscopy

Revealing protein structural evolution is essential for determining its function and interaction with the environment, for understanding disease mechanisms. However, it is challenging for biophysical methods to observe structure evolution, especially at longer distances, limiting their application to few biological systems. Pulsed Dipolar Electron Spin Resonance Spectroscopy (PDS) is powerful method for obtaining such information between the distance ranges of 1 to 10 nm. In PDS, a dipolar signal is acquired from the interaction between a pair of spin labels, from which the distance distribution between them, P(r) may be obtained.

PELDOR Measurements on Nitroxide-Labeled Oligonucleotides.

In the past decades, pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) has emerged as a powerful tool in biophysical chemistry to study the structure, dynamics, and function of biomolecules like oligonucleotides and proteins. Structural information is obtained from PDS methods in form of a distribution of distances between spin centers. Such spin centers can either be intrinsically present paramagnetic metal ions and organic radicals or may be attached to the biomolecule by means of site-directed spin labeling. The most common PDS experiment for probing interspin distances in the nanometer range is pulsed electron-electron double resonance (PELDOR or DEER). In the protocol presented here, we provide a step-by-step workflow on how to set up a PELDOR experiment on a commercially available pulsed EPR spectrometer, outline the data analysis, and highlight potential pitfalls. We suggest PELDOR measurements on nitroxide-labeled oligonucleotides to study the structure of either RNA-cleaving DNAzymes in complex with their RNA targets or modified DNAzymes with different functions and targets, in which deoxynucleotides are substituted by nitroxide-labeled nucleotides.

Fullerene-based triplet spin labels: methodology aspects for pulsed dipolar EPR spectroscopy.

Triplet states of photoexcited organic molecules are promising spin labels with advanced spectroscopic properties for pulsed dipolar electron paramagnetic resonance (PD EPR) spectroscopy. Recently proposed triplet fullerene labels have shown great potential for double electron-electron resonance (DEER) distance measurements as "observer spins" due to a high quantum yield of the triplet state, hyperpolarization and relatively narrow EPR spectra. Here, we demonstrate the applicability of fullerene labels to other PD EPR techniques, such as relaxation induced dipolar modulation enhancement (RIDME) and laser induced magnetic dipolar spectroscopy (LaserIMD). In particular, a specific contaminating signal in LaserIMD experiments was observed, explained and mitigated. Comparative analyses of the signal-to-noise (SNR) ratios were performed for all employed methods. DEER on the fullerene-triarylmethyl pair shows the best performance, which allows state-of-the-art DEER acquisition at 100 nM with a SNR of ∼35 within reasonable 42 hours.

Pulse dipolar EPR for determining nanomolar binding affinities.

Protein interaction studies often require very low concentrations and highly sensitive biophysical methods. Here, we demonstrate that pulse dipolar electron paramagnetic resonance spectroscopy allows measuring dissociation constants in the nanomolar range. This approach is appealing for concentration-limited biomolecular systems and medium-to-high-affinity binding studies, demonstrated here at 50 nanomolar protein concentration.

Uptake of Cell-Penetrating Peptide RL2 by Human Lung Cancer Cells: Monitoring by Electron Paramagnetic Resonance and Confocal Laser Scanning Microscopy.

RL2 is a recombinant analogue of a human κ-casein fragment, capable of penetrating cells and inducing apoptosis of cancer cells with no toxicity to normal cells. The exact mechanism of RL2 penetration into cells remains unknown. In this study, we investigated the mechanism of RL2 penetration into human lung cancer A549 cells by a combination of electron paramagnetic resonance (EPR) spectroscopy and confocal laser scanning microscopy. EPR spectra of A549 cells incubated with RL2 (sRL2) spin-labeled by a highly stable 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl radical were found to contain three components, with their contributions changing with time. The combined EPR and confocal-microscopy data allowed us to assign these three forms of sRL2 to the spin-labeled protein sticking to the membrane of the cell and endosomes, to the spin-labeled protein in the cell interior, and to spin labeled short peptides formed in the cell because of protein digestion. EPR spectroscopy enabled us to follow the kinetics of transformations between different forms of the spin-labeled protein at a minimal spin concentration (3–16 μM) in the cell. The prospects of applications of spin-labeled cell-penetrating peptides to EPR imaging, DNP, and magnetic resonance imaging are discussed, as is possible research on an intrinsically disordered protein in the cell by pulsed dipolar EPR spectroscopy.

Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: CuII-CuII and Nitroxide-Nitroxide Cases.

The study of ever more complex biomolecular assemblies implicated in human health and disease is facilitated by a suite of complementary biophysical methods. Pulse Dipolar electron paramagnetic resonance Spectroscopy (PDS) is a powerful tool that provides highly precise geometric constraints in frozen solution, however the drive towards PDS at physiologically relevant sub-μM concentrations is limited by the currently achievable concentration sensitivity. Recently, PDS using a combination of nitroxide and CuII based spin labels allowed measuring 500 nM concentration of a model protein. Using commercial instrumentation and spin labels we demonstrate CuII-CuII and nitroxide-nitroxide PDS measurements at protein concentrations below previous examples reaching 500 and 100 nM, respectively. These results demonstrate the general feasibility of sub-μM PDS measurements at short to intermediate distances (~1.5 - 3.5 nm), and are of particular relevance for applications where the achievable concentration is limiting.