title: Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy.
abstract: Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data.
authors.affiliation: Departments of Medical Genetics and Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Philipp
authors.initials: P
authors.lastname: Neudecker
authors.forename: Arash
authors.initials: A
authors.lastname: Zarrine-Afsar
authors.forename: Wing-Yiu
authors.initials: WY
authors.lastname: Choy
authors.forename: D Ranjith
authors.initials: DR
authors.lastname: Muhandiram
authors.forename: Alan R
authors.initials: AR
authors.lastname: Davidson
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000818:Animals; D002645:Chickens; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D018909:src Homology Domains
chemical_list: D051076:Proto-Oncogene Proteins c-fyn