title: The PICM chemical scanning method for identifying domain-domain and protein-protein interfaces: applications to the core signaling complex of E. coli chemotaxis.
abstract: The number of known protein structures is growing exponentially (Berman et al., 2000), but the structural mapping of essential domain-domain and protein-protein interaction surfaces has advanced more slowly. It is particularly difficult to analyze the interaction surfaces of membrane proteins on a structural level, both because membrane proteins are less accessible to high-resolution structural analysis and because the membrane environment is often required for native complex formation. The Protein-Interactions-by-Cysteine-Modification (PICM) method is a generalizable, in vitro chemical scanning approach that can be applied to many protein complexes, in both membrane-bound and soluble systems. The method begins by engineering Cys residues on the surface of a protein of known structure, then a bulky probe is coupled to each Cys residue. Next, the effects of both Cys substitution and bulky probe attachment are measured on the assembly and the activity of the target complex. Bulky probe coupling at an essential docking site disrupts complex assembly and/or activity, while coupling outside the site typically has little or no effect. PICM has been successfully applied to the core signaling complex of the E. coli and S. typhimurium chemotaxis pathway, where it has mapped out essential docking surfaces on transmembrane chemoreceptor (Tar) and histidine kinase (CheA) components (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). The approach shares similarities with other important scanning methods like alanine and tryptophan scanning (Cunningham and Wells, 1989; Sharp et al., 1995a), but has two unique features: (1) functional effects are determined for both small volume (Cys) and large volume (bulky probe) side chain substitutions in the same experiment, and (2) nonperturbing positions are identified at which Cys residues and bulky probes can be introduced for subsequent biochemical and biophysical studies, without significant effects on complex assembly or activity.
authors.affiliation: Analytical Sciences, Amgen Inc, Seattle, WA, USA.
authors.forename: Randal B
authors.initials: RB
authors.lastname: Bass
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Miller
authors.forename: Susan L
authors.initials: SL
authors.lastname: Gloor
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D015345:Oligonucleotide Probes / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012486:Salmonella typhimurium / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D015345:Oligonucleotide Probes; D011506:Proteins; D006639:Histidine; D003545:Cysteine