title: Use of site-directed cysteine and disulfide chemistry to probe protein structure and dynamics: applications to soluble and transmembrane receptors of bacterial chemotaxis.
abstract: Site-directed cysteine and disulfide chemistry is broadly useful in the analysis of protein structure and dynamics, and applications of this chemistry to the bacterial chemotaxis pathway have illustrated the kinds of information that can be generated. Notably, in many cases, cysteine and disulfide chemistry can be carried out in the native environment of the protein whether it be aqueous solution, a lipid bilayer, or a multiprotein complex. Moreover, the approach can tackle three types of problems crucial to a molecular understanding of a given protein: (1) it can map out 2 degrees structure, 3 degrees structure, and 4 degrees structure; (2) it can analyze conformational changes and the structural basis of regulation by covalently trapping specific conformational or signaling states; and (3) it can uncover the spatial and temporal aspects of thermal fluctuations by detecting backbone and domain dynamics. The approach can provide structural information for many proteins inaccessible to high-resolution methods. Even when a high-resolution structure is available, the approach provides complementary information about regulatory mechanisms and thermal dynamics in the native environment. Finally, the approach can be applied to an entire protein, or to a specific domain or subdomain within the full-length protein, thereby facilitating a divide-and-conquer strategy in large systems or multiprotein complexes. Rigorous application of the approach to a given protein, domain, or subdomain requires careful experimental design that adequately resolves the structural and dynamical information provided by the method. A full structural and dynamical analysis begins by scanning engineered cysteines throughout the region of interest. To determine 2 degrees structure, the solvent exposure of each cysteine is determined by measuring its chemical reactivity, and the periodicity of exposure is analyzed. To probe 3 degrees structure, 4 degrees structure, and conformational regulation, pairs of cysteines are identified that rapidly form disulfide bonds and that retain function when induced to form a disulfide bond in the folded protein or complex. Finally, to map out thermal fluctuations in a protein of known structure, disulfide formation rates are measured between distal pairs of nonperturbing surface cysteines. This chapter details these methods and illustrates applications to two proteins from the bacterial chemotaxis pathway: the periplasmic galactose binding protein and the transmembrane aspartate receptor.
authors.affiliation: Analytical Sciences, Amgen Inc, Seattle, WA, USA.
authors.forename: Randal B
authors.initials: RB
authors.lastname: Bass
authors.forename: Scott L
authors.initials: SL
authors.lastname: Butler
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Chervitz
authors.forename: Susan L
authors.initials: SL
authors.lastname: Gloor
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D003545:Cysteine