Defining Protein Complexes that Mediate Bacterial Chemotaxis by Pulsed Dipolar ESR Spectroscopy

Abstract

Bacterial chemotaxis is the process whereby cells modulate their flagella-driven motility in response to environmental cues. This widespread behavior relies upon a complex sensory apparatus composed of transmembrane receptors, histidine kinases and coupling proteins to achieve great sensitivity, gain and dynamic range in signal processing. We have applied pulsed-dipolar ESR spectroscopy (PDS) combined with site-directed spin labeling to probe the structures and mechanisms of proteins that compose these regulatory circuits. Inherent symmetries within the protein complexes require interpretation of signals from systems that contain more than two spins. Tikhonov regularization and maximum entropy refinement when combined with model simulation reproduce accurate inter-spin distance distributions from such multiply labeled species. Disulfide crosslinking and disruptive mutagenesis verify component interfaces predicted by PDS. Weak but specific interactions that produce low population aggregates relevant to complex assembly are detected by an approach that relies on magnetic dilution and baseline analysis of dipolar spectra. The resulting PDS-based models capture key architectural features of the receptor kinase arrays and the flagellar motor. Moreover, distance distributions derived from the multi- spin sites reveal changes in conformation and dynamics that accompany kinase activation and motor switching. Application to the chemotaxis system demonstrates how PDS effectively reports on key structural features of transient protein complexes and in doing so fills the resolution gap between other well-established biophysical techniques such as electron microscopy and x-ray crystallography.