Download Conformational dynamics of signaling proteins and ion channels

Conformational dynamics of signaling proteins and ion channels
Gupta, S. and Chance, M.R.
Center for Synchrotron Biosciences, Center for Proteomics and Bioinformatics, Case Western
Reserve University, www.proteomics.case.edu, [email protected]
Radiolytic footprinting and mass spectrometry were used to probe the structure of the inwardly
rectifying potassium channel KirBac 3.1 in its closed and open states. By subjecting protein
solutions to focused synchrotron X-ray beams with millisecond timescale exposures we
modified solvent accessible amino acid aide chains in the membrane pore as well as in the
intercellular domain. Physiologically active preparations of the membrane proteins in detergent
were used to define the structural states of the protein. These modifications were quantified and
identified using high-resolution mass spectrometry. The differences in the extent of such
modifications between the closed and open state reveals the local conformation changes
involving functionally important amino acid residues. The data were analyzed in the context of
the known structure of the closed state where increases in the extent of modification for the
open state provided details of the mechanism of gating and demonstrated a novel method of
probing the dynamics of integral membrane proteins and ion channels.
This result, along with recently published data defining the dynamics of activation of the GPCR
rhodopsin (PNAS, 106:14367, 2009), shows that radiolytic labeling coupled to mass
spectrometry is a unique method to define the structure and dynamics of signaling proteins and
channels in their physiologically active states in detergent or in native membrane preparations.
We present the most recent work defining the dynamics of membrane proteins as well as
introduce novel millisecond timescale O-18 labeling methods to visualize the dynamics of both
bulk water and the dynamics of ordered waters identified crystalographically as being within the
transmembrane segment of rhodopsin.