Supramolecular Approach to Electron Paramagnetic Resonance Distance Measurement of Spin-Labeled Proteins

We
demonstrate a host–guest molecular recognition approach
to advance double electron–electron resonance (DEER) distance
measurements of spin-labeled proteins. We synthesized an iodoacetamide
derivative of 2,6-diazaadamantane nitroxide (DZD) spin label that
could be doubly incorporated into T4 Lysozyme (T4L) by site-directed
spin labeling with efficiency up to 50% per cysteine. The rigidity
of the fused ring structure and absence of mobile methyl groups increase
the spin echo dephasing time (Tm) at temperatures
above 80 K. This enables DEER measurements of distances >4 nm in
DZD-labeled
T4L in glycerol/water at temperatures up to 150 K with increased sensitivity
compared to that of a common spin label such as MTSL. Addition of
β-cyclodextrin reduces the rotational correlation time of the
label, slightly increases Tm, and most
importantly, narrows (and slightly lengthens) the interspin distance
distributions. The distance distributions are in good agreement with
simulated distance distributions obtained by rotamer libraries. These
results provide a foundation for developing supramolecular recognition
to facilitate long-distance DEER measurements at near physiological
temperatures.