Analyzing transmembrane chemoreceptors using in vivo disulfide formation between introduced cysteines.
The sulfhydryl chemistry possible at the thiol group of cysteine provides a very useful tool for probing protein structure and function. The power of site-specific mutagenesis makes it possible to use this tool at essentially any position in a polypeptide sequence. The reactivity of introduced cysteines is often assessed in vitro, using purified proteins or cell extracts. However, it can be particularly informative to probe the protein of interest in vivo, in its native cellular environment. Our laboratory has used in vivo approaches extensively in studies of bacterial transmembrane chemoreceptors, particularly by utilizing disulfide formation between pairs of introduced cysteines to learn about structural organization and mechanisms of function. We have concentrated on experimental conditions in which the cellular system of interest remained functional and thus the protein we were characterizing maintained not only its native structure but also its natural interactions. For this reason, our studies of bacterial transmembrane chemoreceptors using disulfide formation in vivo have focused in large part on cysteines separated from the reducing environment of the cell interior, in transmembrane or periplasmic domains. In this chapter, we discuss the applications and limitation of these approaches as well as the details of experimental manipulations and data analysis.
Lai
Wing-Cheung
WC
Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, USA.
Hazelbauer
Gerald L
GL
D001426:Bacterial Proteins; D001671:Biochemistry / Q000379:methods*; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002628:Chemoreceptor Cells / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry; D008968:Molecular Conformation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors
D001426:Bacterial Proteins; D004220:Disulfides; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D003545:Cysteine; D010100:Oxygen