New Developments in Spin Labels for Pulsed Dipolar EPR

Abstract

Spin labelling is a chemical technique that enables the integration of a molecule containing an unpaired electron into another framework for study. Given the need to understand the structure, dynamics, and conformational changes of biomacromolecules, spin labelling provides a relatively non-intrusive technique and has certain advantages over X-ray crystallography; which requires high quality crystals. The technique relies on the design of binding probes that target a functional group, for example, the thiol group of a cysteine residue within a protein. The unpaired electron is typically supplied through a nitroxide radical and sterically shielded to preserve stability. Pulsed electron paramagnetic resonance (EPR) techniques allow small magnetic couplings to be measured (e.g., <50 MHz) providing information on single label probes or the dipolar coupling between multiple labels. In particular, distances between spin labels pairs can be derived which has led to many protein/enzymes and nucleotides being studied. Here, we summarise recent examples of spin labels used for pulse EPR that serve to illustrate the contribution of chemistry to advancing discoveries in this field.

Probing the Secondary Structure of Membrane Peptides Using 2H-Labeled d10-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy

“…However, with 2 H-labeled side chains with less 2 H atoms and more rigid spin labels such as tetrathiatriarylmethyl (TAM), 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and bifunctional spin label (BSL), more quantitative distance information can be obtained. 39,40 …”

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

“…[11] Importantly,m ost labelling strategies rely on conjugation to cysteinet hiols, [12] which makes orthogonal site-specific labelling problematic.T o overcome this,g enetically encoded labels can be used, though as non-canonical amino-acids, [13] which can be more structurally perturbing than the post-translational modification of natural amino-acids,a nd can also restrict the yield of label incorporation. [11] Importantly,m ost labelling strategies rely on conjugation to cysteinet hiols, [12] which makes orthogonal site-specific labelling problematic.T o overcome this,g enetically encoded labels can be used, though as non-canonical amino-acids, [13] which can be more structurally perturbing than the post-translational modification of natural amino-acids,a nd can also restrict the yield of label incorporation.…”

Nitroxide Spin-Labelling and Its Role in Elucidating Cuproprotein Structure and Function

“…The advantage of these labels is that their ring structure combined with their incorporation into the peptide backbone results in far less conformation freedom compared to nitroxides located on side-chains. Thus, it is more likely that the label accurately reports peptide structure and both mono-and double-labelled peptides have been generated to investigate structures in solution and when membrane bound [15,21]. Labels such as TOAC have been used to study the structure of active peptides such as hormones and neuropeptides; however, these peptides usually bind to cell surface receptors for their activity, and the label can modify receptor binding.…”

“…1), but labels that limit the possible rotamer distributions are being developed [12]. Labelling sites that are not based on cysteine, such as serine and tyrosine have been developed, but do not appear as popular as labels that modify thiols [15].…”

“…The piperidinyl amino acids TOAC (2,2,6,6-tetramethyl-Noxyl-4-amino-4-carboxylic acid) and b-TOAC ( Fig. 1), and the pyrrolidine amino acid POAC (3-amino-1-oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxylic acid) can be incorporated during synthesis [15,21]. The advantage of these labels is that their ring structure combined with their incorporation into the peptide backbone results in far less conformation freedom compared to nitroxides located on side-chains.…”