title: Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes.
abstract: The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 Å) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.
authors.affiliation: Department of Chemistry, ‡Department of Biochemistry and Molecular Biology, and §Program in Molecular and Cellular Biology, University of Massachusetts , Amherst, Massachusetts 01003, United States.
authors.forename: Seena S
authors.initials: SS
authors.lastname: Koshy
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Eyles
authors.forename: Robert M
authors.initials: RM
authors.lastname: Weis
authors.forename: Lynmarie K
authors.initials: LK
authors.lastname: Thompson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002633:Chemotaxis; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry*; D000071677:Histidine Kinase; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D000072236:Methyl-Accepting Chemotaxis Proteins; D008970:Molecular Weight; D055503:Protein Multimerization; D017459:Receptors, Amino Acid / Q000737:chemistry*; D012996:Solutions; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008567:Membranes, Artificial; D000072236:Methyl-Accepting Chemotaxis Proteins; D017459:Receptors, Amino Acid; D012996:Solutions; C031959:aspartic acid receptor; D000071677:Histidine Kinase; C000607608:cheA protein, E coli