posted on 2004-06-15, 00:00authored byLinda Columbus, Wayne L. Hubbell
In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites
in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide
on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from
internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion
of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent,
as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by
comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine
this model, nitroxide side chains were introduced, one at a time, along the GCN4−58 bZip sequence, for
which NMR 15N relaxation experiments have identified a striking gradient of backbone mobility along
the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques
and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and
the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side
chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were
analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation
methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient
of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those
of helices on globular proteins.