Revealing light-induced structural shifts in G-quadruplex-porphyrin complexes: a pulsed dipolar EPR study.

The binding of G-quadruplex structures (G4s) with photosensitizers is of considerable importance in medicinal chemistry and drug discovery due to their promising potential in photodynamic therapy applications. G4s can experience structural changes as a result of ligand interactions and light exposure. Understanding these modifications is essential to uncover the fundamental biological roles of the complexes and optimize their therapeutic potential. The structural diversity of G4s makes it challenging to study their complexes with ligands, necessitating the use of various complementary methods to fully understand these interactions. In this study, we introduce, for the first time, the application of laser-induced dipolar EPR as a method to characterize G-quadruplex DNA complexes containing photosensitizers and to investigate light-induced structural modifications in these systems. To demonstrate the feasibility of this approach, we studied complexes of the human telomeric G-quadruplex (HTel-22) with cationic 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphyrin tetra(p-toluenesulfonate) (TMPyP4). In addition to showcasing a new methodology, we also aimed to provide insights into the mechanisms underlying photoinduced HTel-22/TMPyP4 structural changes, thereby aiding in the advancement of approaches targeting G4s in photodynamic therapy. EPR revealed G-quadruplex unfolding and dimer formation upon light exposure. Our findings demonstrate the potential of EPR spectroscopy for examining G4 complexes with photosensitizers and contribute to a better understanding of G4s' interactions with ligands under light.

Sensitivity optimization in pulse EPR experiments with photo-labels by multiple-echo-integrated dynamical decoupling.

Photo-excited triplet states represent a new class of spin labels in pulse electron paramagnetic resonance (EPR), attracting increasing attention because of their unique spectroscopic properties. Despite certain advantages, the use of photo-labels has also some challenges, e.g. low repetition rates due to technical laser-related limitations and intrinsic properties of the labels. The application of additional pulse trains for multiple refocusing of the electron spin echo and integration of all observed echoes can significantly enhance sensitivity at a given repetition rate. In this work, we demonstrate that the use of Carr-Parcel-Meiboom-Gill (CPMG) blocks followed by multiple echo integration is a promising route for sensitivity gain in pulsed EPR utilizing photo-excited triplet states, including light-induced pulsed dipolar spectroscopy (LiPDS). The reduction of accumulation time by a factor of 5.3 has been achieved using a commercial pulsed EPR spectrometer with the implementation of a CPMG block and an external digitizer. The methodology of using CPMG refocusing with multiple echo integration in light-induced pulsed EPR experiments is discussed, aiding future applications of this approach in LiPDS experiments.

Stability of ZIF-8 Nanoparticles in Most Common Cell Culture Media.

Zeolite imidazolate framework-8 (ZIF-8) is a promising platform for drug delivery, and information regarding the stability of ZIF-8 nanoparticles in cell culture media is essential for proper interpretation of in vitro experimental results. In this work, we report a quantitative investigation of the ZIF-8 nanoparticle's stability in most common cell culture media. To this purpose, ZIF-8 nanoparticles containing sterically shielded nitroxide probes with high resistance to reduction were synthesized and studied using electron paramagnetic resonance (EPR). The degradation of ZIF-8 in cell media was monitored by tracking the cargo leakage. It was shown that nanoparticles degrade at least partially in all studied media, although the degree of cargo leakage varies widely. We found a strong correlation between the amount of escaped cargo and total concentration of amino acids in the environment. We also established the role of individual amino acids in ZIF-8 degradation. Finally, 2-methylimidazole preliminary dissolved in cell culture media partially inhibits the degradation of ZIF-8 nanoparticles. The guidelines for choosing the proper cell culture medium for the in vitro study of ZIF-8 nanoparticles have been formulated.

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.

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.

Two alternative conformations of mRNA in the human ribosome during elongation and termination of translation as revealed by EPR spectroscopy.

The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin-spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.

Application of EPR to porphyrin-protein agents for photodynamic therapy.

Recently, a new type of spin labels based on photoexcited triplet molecules was proposed for nanometer scale distance measurements by pulsed dipolar electron paramagnetic resonance (PD EPR). However, such molecules are also actively used within biological complexes as photosensitizers for photodynamic therapy (PDT) of cancer. Up to date, the idea of using the photoexcited triplets simultaneously as PDT agents and as spin labels for PD EPR has never been employed. In this work, we demonstrate that PD EPR in conjunction with other methods provides valuable information on the structure and function of PDT candidate complexes, exemplified here with porphyrins bound to human serum albumin (HSA). Two distinct porphyrins with different properties were used: amphiphilic meso-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) and water soluble cationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4); HSA was singly nitroxide-labeled to provide a second tag for PD EPR measurements. We found that TMPyP4 locates in a cavity at the center of the four-helix bundle of HSA subdomain IB, close to the interface with solvent, thus being readily accessible to oxygen. As a result, the photolysis of the complex leads to photooxidation of HSA by generated singlet oxygen and causes structural perturbation of the protein. Contrary, in case of mTHPP porphyrin, the binding occurs at the proton-rich pocket of HSA subdomain IIIA, where the access of oxygen to a photosensitizer is hindered. Structural data of PD EPR were supported by other EPR techniques, laser flash photolysis and protein photocleavage studies. Therefore, pulsed EPR on complexes of proteins with photoexcited triplets is a promising approach for gaining structural and functional insights into such PDT agents.

Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins.

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time TM for HSA labeled with OX063 is 6.3 µs at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.

Protein modification by thiolactone homocysteine chemistry: a multifunctionalized human serum albumin theranostic.

As the most abundant protein with a variety of physiological functions, albumin has been used extensively for the delivery of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare spin-labeled albumin-based multimodal imaging probes and therapeutic agents. We report the synthesis of a tamoxifen homocysteine thiolactone derivative and its use in thiol-'click' chemistry to prepare multi-functionalized serum albumin. The released sulfhydryl group of the homocysteine functional handle was labeled with a nitroxide reagent to prepare a spin-labeled albumin-tamoxifen conjugate confirmed by MALDI-TOF-MS, EPR spectroscopy, UV-vis and fluorescent emission spectra. This is the basis for a novel multimodal tamoxifen-albumin theranostic with a significant (dose-dependent) inhibitory effect on the proliferation of malignant cells. The response of human glioblastoma multiforme T98G cells and breast cancer MCF-7 cells to tamoxifen and its albumin conjugates was different in tumor cells with different expression level of ERα in our experiments. These results provide further impetus to develop a serum protein for delivery of tamoxifen to cancer cells.

Unexpected one-pot formation of the 1H-6a,8a-epiminotricyclopenta[a,c,e][8]annulene system from cyclopentanone, ammonia and dimethyl fumarate. Synthesis of highly strained polycyclic nitroxide and EPR study.

The unexpected formation of a highly strained polycyclic amine was observed in a one-pot synthesis from cyclopentanone, dimethyl fumarate and ammonium acetate. This multistep reaction includes 1,3-dipolar cycloaddition of dimethyl fumarate to the cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was easily converted into a sterically shielded polycyclic nitroxide. The EPR spectra and spin relaxation behavior of the nitroxide were studied in solution. The spin relaxation seems well suited for the use as a biological spin label and are comparable with those of cyclic nitroxides with two spirocyclic moieties adjacent to the N-O · group.

Exploring the interactions of short RNAs with the human 40S ribosomal subunit near the mRNA entry site by EPR spectroscopy.

The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human Nav1.4 Channel.

Voltage-gated sodium (NaV) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of NaV1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of NaV1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine micelles. To speed up the assignment of backbone resonances, five selectively 13C,15N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ∼50% of the backbone. Obtained NMR and electron paramagnetic resonance data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps-ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein-protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding ß-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one NaV molecule.

DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

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.

Pulse EPR of Triarylmethyl Probes: A New Approach for the Investigation of Molecular Motions in Soft Matter.

Triarylmethyl (TAM) radicals have become widely used free radicals in the past few years. Their electron spins have long relaxation times and narrow electron paramagnetic resonance (EPR) lines, which make them an important class of probes and tags in biological applications and materials science. In this work, we propose a new approach to characterize librations by means of TAM radicals. The temperature dependence of motional parameter ⟨α2⟩τc, where ⟨α2⟩ is the mean-squared amplitude of librations and τc is their characteristic time, is obtained by comparison of the 1/ Tm phase-relaxation rates at X- and Q-band EPR frequencies. We study three soft matrixes, viz., glassy trehalose and two ionic liquids, using TAMs with optimized relaxation properties OX063D and a dodeca- n-butyl homologue of Finland trityl (DBT). The motional parameters ⟨α2⟩τc obtained using TAMs are in excellent agreement with those obtained by means of nitroxide radicals. At the same time, the new TAM-based approach has (1) greater sensitivity due to the narrower EPR spectrum and (2) greater measuring accuracy and broader temperature range due to longer relaxation times. The developed approach may be fruitfully implemented to probe low-temperature molecular motions of TAM-labeled biopolymers, membrane systems, polymers, molecules in glassy media, and ionic liquids.

Room-temperature distance measurements using RIDME and the orthogonal spin labels trityl/nitroxide.

Electron paramagnetic resonance (EPR) based nanometer distance measurements at ambient temperatures are of particular interest for structural biology applications. The nitroxide spin labels commonly used in EPR reveal relatively short transverse relaxation under these conditions, which limits their use for detecting static dipolar interactions. At the same time, the longitudinal relaxation of nitroxide spin labels is still long enough to allow using them as 'pumped' species in the relaxation induced dipolar modulation enhancement (RIDME) experiment where the detection is carried out on the slower relaxing triarylmethyl (TAM) spin labels. In the present study, we report the first demonstration of room-temperature RIDME distance measurements in nucleic acids using TAM as the slow-relaxing detected species and traditional nitroxide as the fast-relaxing partner spin. Two types of immobilizers, glassy trehalose and the modified silica gel Nucleosil, were used for immobilization of the spin-labeled biomolecules. The room-temperature RIDME-based distance distributions are in good agreement with those measured at 80 K by other techniques. Room-temperature RIDME on the spin pairs trityl/nitroxide may become a useful method for the structural characterization of biomacromolecules and biomolecular complexes at near physiological temperatures.

Structural rearrangements in mRNA upon its binding to human 80S ribosomes revealed by EPR spectroscopy.

The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.

A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures.

Triarylmethyl (trityl, TAM) radicals are a promising class of spin labels for nanometer-scale distance measurements in biomolecules at physiological temperatures. However, to date, existing approaches to site-directed TAM labeling of DNA have been limited to label attachment at the termini of oligonucleotides, thus hindering a majority of demanded applications. Herein, we report a new versatile strategy for TAM attachment at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides, which are then postsynthetically functionalized with TAM. We demonstrate a synthesis of a set of oligonucleotide complexes that are TAM-labeled at internal or terminal sites, as well as the possibility of measuring interspin distances up to ∼5-6 nm at 298 K using double quantum coherence electron paramagnetic resonance (EPR). Implementation of the developed approach strongly broadens the scope of nucleic acids and nucleoprotein complexes available for nanoscale structural EPR studies at room temperatures.

Microscopic rigidity and heterogeneity of ionic liquids probed by stochastic molecular librations of the dissolved nitroxides.

Microscopic molecular organization and heterogeneities in ionic liquids (ILs) are of significant fundamental and applied interest. Although many theoretical studies have been dedicated to this topic, the development of experimental methods for studying such heterogeneities is still in demand. In this work we propose a new approach for the characterization of microscopic rigidity and heterogeneities in ILs using stochastic librations (small angle motions) of the nitroxide radicals as a probe. Stable nitroxides are dissolved in ILs, which are then shock-frozen and investigated using pulse Electron Paramagnetic Resonance (EPR) at variable temperatures. Stochastic molecular librations of nitroxides depend on local rigidity of a medium and manifest themselves in different electron dephasing times across the EPR spectrum. The use of advanced spiro-cyclohexane-substituted nitroxides allows implementation of this approach at temperatures up to ∼150 K and above. Remarkably, we have found that librational motions in ILs arise at much lower temperatures (∼75 K for [Bmim]BF4, [Bmim]PF6, [C10mim]BF4) compared to common organic solvents. This can be rationalized by smaller local rigidity of the medium in ILs, most likely due to localization of nitroxides in low-density heterogeneities formed by non-polar alkyl chains. The structure, size, solubility and other properties of nitroxides are adjustable for particular tasks, therefore the proposed approach can potentially be implemented to probe the rigidity and heterogeneities of any ILs, thus providing vital insights into their molecular-scale self-organization.

Triarylmethyl Radicals: EPR Study of 13C Hyperfine Coupling Constants.

Triarylmethyl (TAM) radicals are widely used in Electron Paramagnetic Resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for in vivo oxymetry. One of the key advantages of TAMs is extremely narrow EPR line, especially in case of deuterated analogues (~5 µT). Another advantage is their slow spin relaxation even at physiological temperatures allowing, in particular, application of pulsed dipolar EPR methods for distance measurements in biomolecules. In this paper a large series of TAM radicals and their deuterated analogues is synthesized, and corresponding spectroscopic parameters including 13C hyperfine constants are obtained for the first time. The negligible dependence of 13C hyperfine constants on solvent, as well as on structure and number of substituents at para-C atoms of aromatic rings, has been found. In addition, we have demonstrated that 13C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER).