Methods in Molecular Biology
Site-directed spin labeling EPR (electron paramagnetic resonance) spectroscopy is a technique used to identify the local conformational changes at a specific residue of interest within a purified protein in response to a ligand. Here, we describe the site-directed spin labeling EPR spectroscopy methodology to monitor changes in the side-chain motion in soluble lipopolysaccharide transport proteins upon the addition of lipopolysaccharide (LPS). A comparison of the spectral overlays of the spin-labeled protein in the absence and presence of LPS provides a qualitative visualization of how LPS binding affects the motion of each spin-labeled site tested within the protein. No change in the spectral lineshapes of a spin-labeled protein in the absence and presence of LPS indicates that the site is not affected by LPS binding, while differences in the spectral lineshapes indicate that LPS does affect the mobility of the spin label side chain within the protein structure. This is a powerful readout of conformational changes at specific residues of interest that can be used to identify a specific site as a reporter of changes induced by ligand binding and to map out the effects of ligand binding through an array of reporter sites within a protein. With the use of AquaStar tubing, protein concentrations as low as 2 μM allow for up to a 100-fold excess of LPS. This methodology may also be applied to other protein-ligand or protein–protein interactions with minor adaptations.