Confinement and Stabilization of Fyn SH3 Folding Intermediate Mimetics within the Cavity of the Chaperonin GroEL Demonstrated by Relaxation-Based NMR.
The interaction of two folding intermediate mimetics of the model protein substrate Fyn SH3 with the chaperonin GroEL, a supramolecular foldase/unfoldase machine, has been investigated by15N relaxation-based nuclear magnetic resonance spectroscopy (lifetime line broadening, dark state exchange saturation transfer, and relaxation dispersion). The two mimetics comprise C-terminal truncations of wild-type and triple-mutant (A39V/N53P/V55L) Fyn SH3 in which the C-terminal strand of the SH3 domain is unfolded, while preserving the remaining domain structure. Quantitative analysis of the data reveals that a mobile state of the SH3 domain confined and tethered within the cavity of GroEL, possibly through interactions with the disordered, methionine-rich C-terminal tail(s), can be detected, and that the native state of the folding intermediate mimetics is stabilized by both confinement within and binding to apo GroEL. These data provide a basis for understanding the passive activity of GroEL as a foldase/unfoldase: the unfolded state, in the absence of hydrophobic GroEL-binding consensus sequences, is destabilized within the cavity because of its larger radius of gyration compared to that of the folding intermediate, while the folding intermediate is stabilized relative to the native state because of exposure of a hydrophobic patch that favors GroEL binding.
Laboratory of Chemical Physics and ‡Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
David S
DS
Libich
Laboratory of Chemical Physics and ‡Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
Vitali
V
Tugarinov
Laboratory of Chemical Physics and ‡Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
Rodolfo
R
Ghirlando
Laboratory of Chemical Physics and ‡Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
G Marius
0000-0003-3809-1027
GM
Clore
D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains
D018834:Chaperonin 60; D009587:Nitrogen Isotopes; D051076:Proto-Oncogene Proteins c-fyn