title: Disulfide-linked bovine beta-lactoglobulin dimers fold slowly, navigating a glassy folding landscape.
abstract: To gain insight into the folding of large proteins, we constructed a bovine beta-lactoglobulin (beta-lg) dimeric mutant, A34C/C121A beta-lg. In the mutant, a free thiol group of wild-type beta-lg at Cys121 was removed and two beta-lg molecules were linked by a disulfide bridge through Cys34 created at the dimer's interface. Under strongly native conditions at low concentrations of urea, the refolding yield of A34C/C121A beta-lg was low when monitored by heteronuclear NMR spectroscopy. However, under marginally native conditions, the yield improved notably, although the refolding was still slow. H-D exchange pulse labeling monitored using heteronuclear NMR spectroscopy indicated that A34C/C121A beta-lg forms a folding intermediate similar to monomeric C121A beta-lg in spite of its slow folding. These results indicate that the rapid formation of folding intermediates driven by local interactions occurs in a manner independent of the molecular size and that, if the non-native interactions are too strong, the kinetic trap is set, leading to a glasslike misfolded state. The results suggest the important roles of marginal stability and pathways in making the folding of large proteins possible.
authors.affiliation: Institute for Protein Research, Osaka University, and CREST, Japan Science and Technology Agency, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Yagi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kameda
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Sakurai
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nishimura
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
chemical_list: D004220:Disulfides; D007782:Lactoglobulins; D014508:Urea