Probing Orientations of Single Fluorescent Labels on a Peptide Reversibly Binding to the Human Delta-Opioid Receptor

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Abstract
We report the first in-depth study of single-molecule polarization behavior of a species that is undergoing reversible binding with its biological receptor. We examine the utility of the information in single-molecule polarization measurements for investigations of binding. The human δ-opioid receptor, which is a G protein-coupled receptor, was incorporated into a supported lipid bilayer. A Cy3 label was covalently attached by a hydrophilic linker to a peptide agonist, Deltorphin II (5,6 Ile-Ile). The fluorescence excitation was alternated between s- and p-polarization using a microscope having the capability of total internal reflectance fluorescence (TIRF) excitation. The polarization behavior reveals that nonspecific binding events for this system give emission that is mostly s-polarized, while binding to the receptor gives emission that has a strong component of p-polarization. The results show that a high signal-to-noise ratio is achievable with single-molecule polarization measurements. The experiment detected 37 binding events of short duration (<30 s) and 35 binding events of long duration (from 30 s to 500 s). The polarization studies indicate that the receptors in the bilayer do not freely rotationally diffuse in the plane of the bilayer when the peptide is bound. The system exhibits two types of polarization behavior. One type has the dye label with fixed orientation, which sometimes abruptly switches. The other type has the dye orientation continuously fluctuating over time, typically exhibiting occasional periods of fixed orientation. For a long binding event of fixed orientation, it is established through analysis of the variance that the orientation actually is fluctuating through a range of angles on the order of 6°. It is shown that precise measurements of reorientation are achievable, with a detection limit of 1.3° for a typical single-molecule signal.