Double Quantum Coherence ESR at Q-Band Enhances the Sensitivity of Distance Measurements at Submicromolar Concentrations

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

“…This would likely demand concentrations higher than those achieved here. Together with recent advances in hardware, , we predict that single-digit nanomolar PDS distance measurements will soon be within reach in favorable cases. This will enable new science, particularly with respect to concentration-limited systems.…”

“…To enable direct comparison to some of the previous benchmarking studies, , dHis-CuNTA and SLIM were grafted onto the Streptococcus sp. group G protein G, B1 domain (GB1), an established model protein extensively used for EPR studies. ,,,,,,,,, Here, the GB1 construct I6C/K28H/Q32H was used, , bearing the dHis motif for coordination to CuNTA in an α-helix and a cysteine residue for SLIM labeling in a β-sheet (Figure ).…”

“…Recent developments in electron paramagnetic resonance (EPR) spectroscopy have firmly established PDS in the toolbox for structural biology, due to its accuracy, reproducibility, and concentration sensitivity, and by providing access to full conformational ensembles of biomolecules. − PDS methods such as pulsed electron–electron double resonance (PELDOR or DEER), − relaxation-induced dipolar modulation enhancement (RIDME), − and double quantum coherence (DQC) ,, provide distance distributions in the nanometer range (from ∼2 to ∼16 nm) − and have become important tools for studying complex biomolecular systems. , …”

“…In most cases, paramagnetic centers are introduced via site-directed spin-labeling (SDSL) of specific residues, usually employing stable organic radicals such as nitroxides ,− or trityls ,, or paramagnetic metal ions such as Gd III , ,,− Mn II , or Cu II . − In most cases, SDSL involves stable radicals introduced via cysteine-specific chemistry, where the label-specific length of the linker contributes degrees of freedom to the dynamics of the protein, which artificially broadens distance distributions or allows only distinct label conformations that could make interpretation of distance distributions ambiguous. , Additionally, different spin-labels may lead to different degrees of perturbation of the native structure due to their size and interactions with protein residues, e.g., trityl labels. Another approach uses Cu II complexed with nitrilotriacetic acid (CuNTA) coordinated to a site-specifically introduced double-histidine (dHis) motif (dHis-CuNTA), posing more stringent requirements on the labeling site but yielding very narrow distance distributions due to the rigidity of this labeled side chain. ,,− CuNTA increases specificity to dHis sites compared to free Cu II in solution, which has a stronger propensity for unspecific binding. ,, Depending on the secondary structural elements (α-helix or β-sheet), dissociation constants ( K d ) were determined to be on the order of 10 –5 to 10 –7 under EPR conditions. , An advantage of using spectroscopically orthogonal labels is that binding sites can be saturated by an excess of CuNTA without overlap with the detected signal .…”

“…In most cases, paramagnetic centers are introduced via site-directed spin-labeling (SDSL) of specific residues, usually employing stable organic radicals such as nitroxides 1 , 33 − 35 or trityls 3 , 23 , 36 or paramagnetic metal ions such as Gd III , 21 , 34 , 36 − 39 Mn II , 40 or Cu II . 41 − 44 In most cases, SDSL involves stable radicals introduced via cysteine-specific chemistry, where the label-specific length of the linker contributes degrees of freedom to the dynamics of the protein, which artificially broadens distance distributions or allows only distinct label conformations that could make interpretation of distance distributions ambiguous.…”