Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions. Methods Mol Biol 2022;2548:83-96
Date
09/24/2022Pubmed ID
36151493Pubmed Central ID
PMC10765611DOI
10.1007/978-1-0716-2581-1_6Scopus ID
2-s2.0-85138460647 (requires institutional sign-in at Scopus site)Abstract
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.
Author List
Schultz KM, Klug CSAuthors
Candice S. Klug PhD Professor in the Biophysics department at Medical College of WisconsinKathryn M. Schultz Research Scientist I in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Carrier ProteinsElectron Spin Resonance Spectroscopy
Ligands
Lipopolysaccharides
Proteins
Spin Labels
