Abstract
The
study of complex biomolecular assemblies implicated in human health and disease
is increasingly performed under native conditions. Pulse Dipolar Electron
paramagnetic resonance (PDEPR) spectroscopy is a powerful tool that provides
highly precise geometric constraints in frozen solution, however the drive
towards in cellulo EPR is limited by the currently achievable
concentration sensitivity in the low μM regime. Achieving PDEPR at physiologically relevant
sub-μM concentrations is currently very challenging. Recently, relaxation
induced dipolar modulation enhancement (RIDME) measurements using a combination
of nitroxide and double-histidine CuII based spin labels allowed
measuring 500 nM concentration of a model protein. Herein, we demonstrate CuII-CuII
RIDME and nitroxide-nitroxide PELDOR measurements down to 500 and 100 nM protein
concentration, respectively. This is possible using commercial instrumentation
and spin labels. These results herald a transition towards routine sub-μM PDEPR
measurements at short to intermediate distances (~1.5-3.5 nm), without the necessity
of specialized instrumentation or spin-labelling protocols, particularly
relevant for applications in near physiological conditions.
Supplementary materials
Title
GB1 nMChemRxiv SI
Description
Actions