TY - JOUR
T1 - Protein footprinting in a complex milieu
T2 - Identifying the interaction surfaces of the chemotaxis adaptor protein chew
AU - Underbakke, Eric S.
AU - Zhu, Yimin
AU - Kiessling, Laura L.
N1 - Funding Information:
This research was supported by the National Institutes of Health ( GM55984 ). E.S.U. was supported by the Molecular Biosciences Training Grant Program ( GM07215 ). We thank J. S. Parkinson for helpful comments on the manuscript.
PY - 2011/6/17
Y1 - 2011/6/17
N2 - Characterizing protein-protein interactions in a biologically relevant context is important for understanding the mechanisms of signal transduction. Most signal transduction systems are membrane associated and consist of large multiprotein complexes that undergo rapid reorganization-circumstances that present challenges to traditional structure determination methods. To study protein-protein interactions in a biologically relevant complex milieu, we employed a protein footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. ICAT reagents are valuable tools for proteomics. Here, we show their utility in an alternative application-they are ideal for protein footprinting in complex backgrounds because the affinity tag moiety allows for enrichment of alkylated species prior to analysis. We employed a water-soluble ICAT reagent to monitor cysteine accessibility and thereby to identify residues involved in two different protein-protein interactions in the Escherichia coli chemotaxis signaling system. The chemotaxis system is an archetypal transmembrane signaling pathway in which a complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends on the adaptor protein CheW, which mediates a functionally important bridging interaction between transmembrane receptors and histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large multidomain histidine kinase CheA, as well as with the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA-Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite CheW-Tsr surface for the recruitment of the signaling kinase to the chemoreceptor complex.
AB - Characterizing protein-protein interactions in a biologically relevant context is important for understanding the mechanisms of signal transduction. Most signal transduction systems are membrane associated and consist of large multiprotein complexes that undergo rapid reorganization-circumstances that present challenges to traditional structure determination methods. To study protein-protein interactions in a biologically relevant complex milieu, we employed a protein footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. ICAT reagents are valuable tools for proteomics. Here, we show their utility in an alternative application-they are ideal for protein footprinting in complex backgrounds because the affinity tag moiety allows for enrichment of alkylated species prior to analysis. We employed a water-soluble ICAT reagent to monitor cysteine accessibility and thereby to identify residues involved in two different protein-protein interactions in the Escherichia coli chemotaxis signaling system. The chemotaxis system is an archetypal transmembrane signaling pathway in which a complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends on the adaptor protein CheW, which mediates a functionally important bridging interaction between transmembrane receptors and histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large multidomain histidine kinase CheA, as well as with the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA-Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite CheW-Tsr surface for the recruitment of the signaling kinase to the chemoreceptor complex.
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U2 - 10.1016/j.jmb.2011.03.040
DO - 10.1016/j.jmb.2011.03.040
M3 - Article
C2 - 21463637
AN - SCOPUS:79957741036
SN - 0022-2836
VL - 409
SP - 483
EP - 495
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -