Rotational Coupling in Methyl-Tunneling Electron Spin Echo Envelope Modulation

Coherence between tunnel-split states of a methyl quantum rotor can be generated and observed in stimulated and spin-locked echo experiments, if hyperfine coupling of a nearby electron spin to the methyl protons breaks C$$_3$$ 3 symmetry and is of the same order of magnitude as the tunnel splitting. Here, we consider the case of two methyl groups bound to the same sp$$^{3}$$ 3 -hybridized atom, which is important in the context of common nitroxide spin labels. For a simple form of the rotor-rotor coupling Hamiltonian, we provide an approach that allows for density operator computations of this system with 1152 quantum states with moderate computational effort. We find that, in the regime where the ratio between rotor-rotor coupling and rotational barrier is much smaller than unity, three-pulse ESEEM and hyperfine-decoupled ESEEM depend only on the tunnel splitting, but not on this ratio. This finding may simplify the treatment of tunnel-induced electron decoherence in systems where the methyl groups are bound to sp$$^{3}$$ 3 -hybridized atoms.

DEER and RIDME Measurements of the Nitroxide-Spin Labelled Copper-Bound Amine Oxidase Homodimer from Arthrobacter Globiformis

In the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin–spin distances at nanometre-scale by measuring dipole–dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO.

Reconciling Membrane Protein Simulations with Experimental DEER Spectroscopy Data

Spectroscopy experiments are crucial to study membrane proteins for which traditional structure determination methods still prove challenging. Double electron-electron resonance (DEER) spectroscopy experiments provide protein residue-pair distance distributions that are indicative of their conformational heterogeneity. Atomistic molecular dynamics (MD) simulations are another tool that have proved vital to study the structural dynamics of membrane proteins such as to identify inward-open, occluded, and outward-open conformations of transporter membrane proteins, among other partially open or closed states of the protein. Yet, studies have reported that there is no direct consensus between distributional data from DEER experiments and MD simulations, which has challenged validation of structures obtained from long-timescale simulations and using simulations to design experiments. Current coping strategies for comparisons rely on heuristics, such as mapping nearest matching peaks between two ensembles or biased simulations. Here we examine the differences in residue-pair distance distributions arising due to choice of membrane around the protein and covalent modification of a pair of residues to nitroxide spin labels in DEER experiments. Through comparing MD simulations of two proteins, PepTSo and LeuT - both of which have been characterized using DEER experiments previously - we show that the proteins’ dynamics are similar despite the choice of the detergent micelle as a membrane mimetic in DEER experiments. On the other hand, covalently modified residues show slight local differences in their dynamics and a huge divergence when the spin labels’ anointed oxygen atom pair distances are measured rather than protein backbone distances. Given the computational expense associated with pairwise MTSSL labeled MD simulations, we examine the use of biased simulations to explore the conformational dynamics of the spin labels only to reveal that such simulations alter the underlying protein dynamics. Our study identifies the main cause for the mismatch between DEER experiments and MD simulations and will accelerate developing potential mitigation strategies to improve simulation observables match with DEER spectroscopy experiments.

Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

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.