title: Disulfide formation as a probe of folding in GroEL-GroES reveals correct formation of long-range bonds and editing of incorrect short-range ones.
abstract: The chaperonin GroEL assists protein folding by binding nonnative forms through exposed hydrophobic surfaces in an open ring and mediating productive folding in an encapsulated hydrophilic chamber formed when it binds GroES. Little is known about the topology of nonnative proteins during folding inside the GroEL-GroES cis chamber. Here, we have monitored topology employing disulfide bond formation of a secretory protein, trypsinogen (TG), that behaves in vitro as a stringent, GroEL-GroES-requiring substrate. Inside the long-lived cis chamber formed by SR1, a single-ring version of GroEL, complexed with GroES, we observed an ordered formation of disulfide bonds. First, short-range disulfides relative to the primary structure formed, both native and nonnative. Next, the two long-range native disulfides that "pin" the two beta-barrel domains together formed. Notably, no long-range nonnative bonds were ever observed, suggesting that a native-like long-range topology is favored. At both this time and later, however, the formation of several medium-range nonnative bonds mapping to one of the beta-barrels was observed, reflecting that the population of local nonnative structure can occur even within the cis cavity. Yet both these and the short-range nonnative bonds were ultimately "edited" to native, as evidenced by the nearly complete recovery of native TG. We conclude that folding in the GroEL-GroES cavity can favor the formation of a native-like topology, here involving the proper apposition of the two domains of TG; but it also involves an ATP-independent conformational "editing" of locally incorrect structures produced during the dwell time in the cis cavity.
authors.affiliation: Department of Genetics, Yale School of Medicine, New Haven, CT 06510, USA.
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Park
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Fenton
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Horwich
mesh_terms: D000818:Animals; D002417:Cattle; D018835:Chaperonin 10 / Q000737:chemistry*; D018834:Chaperonin 60 / Q000737:chemistry*; D004220:Disulfides; D013058:Mass Spectrometry; D011487:Protein Conformation; D017510:Protein Folding; D014362:Trypsinogen / Q000737:chemistry / Q000378:metabolism
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D004220:Disulfides; D014362:Trypsinogen