Measuring Nanometer Distances in Proteins and Rigid Rulers between 19F and Gd3+ by Integration of 19F-ENDOR Signal Intensities

Abstract

¹⁹F ENDOR is emerging as a powerful tool in structural biology for measuring distances in proteins labeled with ¹⁹F and a paramagnetic tag. Due to spin–spin relaxation and line width limitations, it has been difficult to determine intertag distances larger than about 15 Å. Using a set of geometrically well-defined rulers and spin-labeled proteins, we show that ¹⁹F–Gd³⁺ distances up to 20 Å can be accessed by integrating the intensity of the ENDOR spectrum, with distances approaching 30 Å potentially in reach as well. This method is robust when the intensities are scaled to a known reference, and provides scope for nanometer-scale triangulation of the coordinates of a ligand in a protein–ligand complex.