Advances in electron paramagnetic resonance through synthetic chemistry

Abstract

Electron paramagnetic resonance spectroscopy (EPR) is one of the most powerful and versatile tools for investigating the local structure and dynamics of paramagnetic substances. In this work, dipolar spectroscopy (PDS) and hyper fine spectroscopy (HFS) are used to investigate the binding and coordination of metal ions such as gadolinium (Gd(III)) and copper (Cu(II)) to de novo coiled-coil (Cc) peptides to characterise their local environment, and probe the nature of the binding site. This work highlights the versatility of designer miniature protein frameworks, which give access to unique and functional binding sites.

This flexibility is seen through investigations of the miniature protein Cc MB1-2, presenting the characterisation of a novel oxophilic Cu(II) binding site, within a protein scaffold and a series of Cc's based on modi cation of this binding site. Mutation and translation of this site shows how the EPR parameters of the Cu(II) ion can be tuned depending on the nature of the available amino acids.

High-spin EPR spin labels such as Gd(III) are of interest owing to their stability within cellular environments. However, PDS measurements using Gd(III) are known to be inaccurate at short distances (<4 nm) when excitation of the central transition (CT) often leads to unwanted excitation of flip-flop transitions causing spectral distortions. New PDS techniques are detailed that can provide high-sensitivity, artefact free data in high-spin systems by avoiding the CT at W-band. RIDME measurements show when measuring short-distances, RIDME is preferred over DEER and provides higher quality data. Amplitude and phase modulated chirp pulses are also compared against standard rectangular pulses in a variety of PDS experiments. Finally, translation of a double Gd(III) binding Cc with known distances, shows promise as a ruler system for PDS measurements, moving away from purely synthetic model rulers whose flexibility do not mirror those of native biological systems.