Spectroscopically Orthogonal Labelling to Disentangle Site-Specific Nitroxide Label Distributions

Abstract

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

“…For samples with a protein concentration of <100 nM, recording the reference traces became unfeasible due to the required extended averaging times and was therefore done only for concentrations of ≥100 nM. As observed previously, , modulation depths were slightly higher for the vtRIDME than for the ctRIDME. Deconvolution reduced the observed modulation depths for both ct- and vtRIDME measurements as expected.…”

“…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 . Furthermore, the dHis-CuNTA labeling has been shown to be robust against competing ligands and to retain its high affinity binding over a wide pH range, thus demonstrating biologically relevant compatibility .…”

“…(B) Visualization of the labeled GB1 construct, with modeled dHis-CuNTA and SLIM rotamers shown as sticks. (C) Simulated distance distribution obtained from Colab running mtsslWizard for bipedal labels. ,, …”

“…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.…”

“…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. 35 , 42 , 47 − 50 CuNTA increases specificity to dHis sites compared to free Cu II in solution, which has a stronger propensity for unspecific binding. 42 , 47 , 51 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.…”