title: Visualizing side chains of invisible protein conformers by solution NMR.
abstract: Sparsely populated and transiently formed protein conformers can play key roles in many biochemical processes. Understanding the structure function paradigm requires, therefore, an atomic-resolution description of these rare states. However, they are difficult to study because they cannot be observed using standard biophysical techniques. In the past decade, NMR methods have been developed for structural studies of these elusive conformers, focusing primarily on backbone (1)H, (15)N and (13)C nuclei. Here we extend the methodology to include side chains by developing a (13)C-based chemical exchange saturation transfer experiment for the assignment of side-chain aliphatic (13)C chemical shifts in uniformly (13)C labeled proteins. A pair of applications is provided, involving the folding of β-sheet Fyn SH3 and α-helical FF domains. Over 96% and 89% of the side-chain (13)C chemical shifts for excited states corresponding to the unfolded conformation of the Fyn SH3 domain and a folding intermediate of the FF domain, respectively, have been obtained, providing insight into side-chain packing and dynamics.
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bouvignies
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Pramodh
authors.initials: P
authors.lastname: Vallurupalli
authors.affiliation: Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemistry, The University of Toronto, Toronto, Ontario M5S 1A8, Canada; Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5S 1A8, Canada. Electronic address: kay@pound.med.utoronto.ca.
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000818:Animals; D002645:Chickens; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D017510:Protein Folding; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D051076:Proto-Oncogene Proteins c-fyn
keywords: 2D; BMRB; Biological Magnetic Resonance Bank; CEST; CPMG; Carr–Purcell–Meiboom–Gill; FID; chemical exchange saturation transfer; chemical shifts; conformationally excited states; free induction decay; protein side chains; two-dimensional