SLIM: A Short‐Linked,Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm , and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.     

Photochemical Reactions and Photoinduced Electron‐Transfer Processes in Liquids,Frozen Solutions,and Proteins as Studied by Multifrequency Time‐Resolved EPR Spectroscopy

In this overview, modern multifrequency EPR spectroscopy, in particular at high magnetic fields, is shown to provide detailed information about structure, motional dynamics, and spin chemistry of transient radicals and radical pairs occurring in photochemical reactions. Examples discussed comprise photochemical reactions in liquid solution and light‐initiated electron transfer processes both in biomimetic donor–acceptor model systems in frozen solution or liquid crystals and in natural photosynthetic‐reaction‐center protein complexes. The transient paramagnetic states exhibit characteristic electron polarization (CIDEP) effects. They contain valuable information about structure and dynamics of the transient reaction intermediates. Moreover, they are exploited for signal enhancement. Continuous‐wave (cw) and pulsed versions of time‐resolved high‐field EPR spectroscopy, such as cw‐transient‐EPR (TREPR) and pulsed‐electron‐spin‐echo (ESE) experiments, are compared with respect to their advantages and limitations for the specific system under study. For example, W‐band (95‐GHz) TREPR spectroscopy in conjunction with a continuous‐flow system for light‐generated short‐lived transient spin‐polarized radicals of organic photoinitiators in solution was performed with a time resolution of 10 ns. The increased Boltzmann polarization at high fields even allows detection of transient radicals without CIDEP effects. This enables one to determine initial radical polarization contributions as well as radical‐addition reaction constants. Another example of the power of combined X‐band and W‐band TREPR spectroscopy is given for the complex electron‐transfer and spin dynamics of covalently linked porphyrin–quinone as well as Watson–Crick base‐paired porphyrin–dinitrobenzene donor–acceptor biomimetic model systems. Furthermore, W‐band ESE experiments on the spin‐correlated coupled radical pair in reaction centers of the purple photosynthetic bacterium Rb. sphaeroides reveal details of distance and orientation of the pair partners in their charge‐separated transient state. The results are compared with those of the ground‐state P₈₆₅Q_A. The high orientation selectivity of high‐field EPR provides single‐crystal‐like information even from disordered frozen‐solution samples. The examples given demonstrate that high‐field EPR adds substantially to the capability of ‘classical’ spectroscopic and diffraction techniques for determining structure–dynamics–function relations of biochemical systems, since transient intermediates can be observed in real time in their working states on biologically relevant time scales.     

In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

Post‐synthetic Spin‐Labeling of RNA through Click Chemistry for PELDOR Measurements

Site‐directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron‐electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here d? . We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW‐EPR as well as PELDOR we analyzed and compared the influence of d? and Çm on a self‐complementary RNA duplex. Our results demonstrate that the conformational diversity of d? is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation‐selective PELDOR time traces of the d? ‐labeled RNA duplex.     

SLIM: A Short-Linked,Highly Redox-Stable Trityl Label for High-Sensitivity In-Cell EPR Distance Measurements

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site-directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in-cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide-functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm , and shows high stability against reduction. Using this label, the guanine-nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.     

Effects of cation siting and spin–spin interactions on the electron paramagnetic resonance (EPR) of Cu exchanged X Faujasite zeolite

Copper(II) exchanged Na X Faujasite zeolite was cation exchanged at levels from one Cu(II) in 30 unit cells (0.033 Cu(II)/UC) to 38 Cu(II) per unit cell (38 Cu/UC) and was examined by continuous wave and two-pulse and three-pulse electron paramagnetic resonance (EPR) at temperatures from 10 K to 300 K. In this work exchange of Cu²⁺ into X Faujasite zeolite is shown by EPR spectral and pulsed EPR relaxation measurements to begin into site I′, where it lies coordinated to a hexagonal prism face with Si:Al ratios of predominantly 4:2 and 5:1. Spin–spin interactions influence EPR g-value averaging, spin–spin relaxation, and spin spectral diffusion in a manner highly dependent on Cu exchange. Spin–lattice relaxation is relatively independent of exchange. The marked increase observed in spin–spin relaxation and g-value averaging at 8 Cu/UC and an effective Cu–Cu distance of 1.2 nm can be understood in terms of filling sodalite cages with an average of 1 Cu²⁺ each.     

Novel Pulsed Electron Paramagnetic Resonance Techniques for the Studies of Structure and Dynamics of Photo‐excited Triplet State of Organic Molecules: A Professional Journey

We present a few novel pulsed electron paramagnetic resonance techniques developed in our laboratory for the studies of structure and dynamics of the photo‐excited triplet state of organic molecules. We discuss many aspects of these new techniques and the significances of these measurements: (1) enhancing NMR signal intensity by dynamic nuclear polarization ‐ integrated solid effect, (2) performing magnetic resonance in zero‐field and low‐field by pulsed microwave, (3) mapping molecular motion of organic crystals by pulsed zero‐field and low‐field experiments, (4) probing spin dynamics at level anti‐crossing by fast field switching, (5) measuring hyperfine interaction by electron spin echo envelop modulation and spin‐echo electron nuclear double resonance and (6) detecting spin dynamics, nuclear quantum oscillation, entanglements and new avenues for quantum computer. We have employed the highly electron spin polarized pentacene triplet state as the model system in all of our pulsed EPR experiments. We performed most of our experiments at room temperature. The goals of our studies are aiming to improve spin detectability, to probe molecular dynamics, to determine electronic structures, to measure molecular interaction and motion, and to examine quantum coherence and oscillation which may yield new avenues in the applications of pulsed EPR techniques to quantum computer.     

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near‐room temperature. This approach is demonstrated using fullerene–nitroxide and fullerene–triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide‐labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.     

Discrepancy between the Spin Distribution and the Magnetic Ground State for a Triaminoxyl Substituted Triphenylphosphine Oxide Derivative

The magnetic interaction and spin transfer via phosphorus have been investigated for the tri‐tert‐butylaminoxyl para‐substituted triphenylphosphine oxide. For this radical unit, the conjugation existing between the π* orbital of the NO group and the phenyl π orbitals leads to an efficient delocalization of the spin from the radical to the neighboring aromatic ring. This has been confirmed by using fluid solution high‐resolution EPR and solid state MAS NMR spectroscopy. The spin densities located on the atoms of the molecule could be probed since ¹H, ¹³C, ¹⁴N, and ³¹P are nuclei active in NMR and EPR, and lead to a precise spin distribution map for the triradical. The experimental investigations were completed by a DFT computational study. These techniques established in particular that spin density is located at the phosphorus (ρ=?15×10^?3 au), that its sign is in line with the sign alternation principle and that its magnitude is in the order of that found on the aromatic C atoms of the molecule. Surprisingly, whereas the spin distribution scheme supports ferromagnetic interactions among the radical units, the magnetic behavior found for this molecule revealed a low‐spin ground state characterized by an intramolecular exchange parameter of J=?7.55 cm^?1 as revealed by solid state susceptibility studies and low temperature EPR. The X‐ray crystal structures solved at 293 and 30 K show the occurrence of a crystallographic transition resulting in an ordering of the molecular units at low temperature.     

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with Cu^II spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent Cu^II coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (K_D) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm Cu^II K_Ds under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled Cu^II–nitroxide systems using a commercial Q‐band EPR instrument.     

Light‐Induced Porphyrin‐Based Spectroscopic Ruler for Nanometer Distance Measurements

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin‐labeling method based on the photoexcited triplet state (S=1). Four‐pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N‐terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8–8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR‐derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)–porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.     

Spin Crossover and Valence Tautomerism in Neutral Homoleptic Iron Complexes of Bis(pyridylimino)isoindolines

Homoleptic iron complexes of six bis(pyridylimino)isoindoline (bpi) ligands with different substituents (H, Me, Et, tBu, OMe, NMe₂) at the 4‐positions of the pyridine moieties have been prepared and studied with regard to temperature‐dependent spin and redox states by a combination of ⁵⁷Fe Mössbauer spectroscopy, SQUID magnetometry, single‐crystal X‐ray diffraction analysis, X‐band EPR, and ¹H NMR spectroscopy. While the H‐, methyl‐, and ethyl‐substituted complexes remain in a pure high‐spin state irrespective of the temperature, the 4‐tert‐butyl‐substituted derivative shows spin‐crossover behavior. The methoxy‐ and dimethylamino‐substituted compounds were found to easily undergo oxidation. In the crystalline state, valence tautomeric behavior was observed for the methoxy derivative as a thermally activated charge‐transfer transition, accompanied by a spin crossover above 200 K. The valence tautomerism leads to a chelate with one of the bpi ligands as a dianion radical L^2?. and with an effective spin of S=2.     

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.     

gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis,EPR Characterization,Rotamer Libraries and Biocompatibility

The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.     

Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.     

Studies of a Large Odd‐Numbered Odd‐Electron Metal Ring: Inelastic Neutron Scattering and Muon Spin Relaxation Spectroscopy of Cr8Mn

The spin dynamics of Cr₈Mn, a nine‐membered antiferromagnetic (AF) molecular nanomagnet, are investigated. Cr₈Mn is a rare example of a large odd‐membered AF ring, and has an odd‐number of 3d‐electrons present. Odd‐membered AF rings are unusual and of interest due to the presence of competing exchange interactions that result in frustrated‐spin ground states. The chemical synthesis and structures of two Cr₈Mn variants that differ only in their crystal packing are reported. Evidence of spin frustration is investigated by inelastic neutron scattering (INS) and muon spin relaxation spectroscopy (μSR). From INS studies we accurately determine an appropriate microscopic spin Hamiltonian and we show that μSR is sensitive to the ground‐spin‐state crossing from S=1/2 to S=3/2 in Cr₈Mn. The estimated width of the muon asymmetry resonance is consistent with the presence of an avoided crossing. The investigation of the internal spin structure of the ground state, through the analysis of spin‐pair correlations and scalar‐spin chirality, shows a non‐collinear spin structure that fluctuates between non‐planar states of opposite chiralities.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

Electron spin relaxation time of Ni(II) ion in hexapyrazole zinc(II) dinitrate at 300 K

Understanding the electron spin relaxation properties of paramagnetic species is a fundamental requirement to use them as a probe to measure distances between sites in biomolecules by electron paramagnetic resonance (EPR) spectroscopy. Even though Ni(II) ion is an essential trace element for many species, relaxation properties are not well understood. Herein, the polycrystalline sample of Ni(II) ion magnetically diluted in Zn(Pyrazole)₆(NO₃)₂ (Ni/ZPN) has been studied in detail by EPR spectroscopy to explore the electron spin relaxation time. Progressive continuous-wave (CW) EPR power saturation study on Ni/ZPN at 300 K yielded 907 mW as the P_1/2 value. The cavity constant (K_Q) has been calculated using tempol in PVA-BA glass matrix and the product of electron spin-lattice relaxation time (T₁) and spin–spin relaxation time (T₂) for Ni/ZPN at 300 K has been reported for the first time.     

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for ¹H and ¹³C (¹H→¹³C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for ¹H and 13.9±0.5 for ¹³C (direct polarization).