title: Analysis of protein structure in intact cells: crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor.
abstract: Oxidative crosslinking of cysteines introduced by site-specific mutagenesis is a powerful tool for structural analysis of proteins, but the approach has been limited to studies in vitro. We recently reported that intact cells of Escherichia coli could be treated with Cu(II)-(o-phenanthroline)3 or molecular iodine in a way that left unperturbed flagellar function or general chemotactic response, yet crosslinks were quantitatively formed between select cysteines in adjoining transmembrane helices of chemoreceptor Trg. This suggested that oxidative crosslinking might be utilized for structural analysis in vivo. Thus, we used our comprehensive collection of Trg derivatives, each containing a single cysteine at one of the 54 positions in the two transmembrane segments of the receptor monomer to characterize patterns of crosslinking in vivo and in vitro for this homodimeric protein. We found that in vivo crosslinking compared favorably as a technique for structural analysis with the more conventional in vitro approach. Patterns of crosslinking generated by oxidation treatments of intact cells indicated extensive interaction of transmembrane segment 1 (TM1) with its homologous partner (TM1') in the other subunit and a more distant placement of TM2 and TM2', the same relationships identified by crosslinking in isolated membranes. In addition, the same helical faces for TM1-TM1' interaction and TM2-TM2' orientation were identified in vivo and in vitro. The correspondence of the patterns also indicates that structural features identified by analysis of in vitro crosslinking are relevant to the organization of the chemoreceptor in its native environment, the intact, functional cell. It appears that the different features of the two functionally benign treatments used for in vivo oxidations can provide insights into protein dynamics.
authors.affiliation: Department of Genetics and Cell Biology, Washington State University, Pullman 99164-4660, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Hughson
authors.forename: G F
authors.initials: GF
authors.lastname: Lee
authors.forename: G L
authors.initials: GL
authors.lastname: Hazelbauer
mesh_terms: D001426:Bacterial Proteins; D002628:Chemoreceptor Cells / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D003545:Cysteine