(13)CHD2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.
An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of (13)CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this (13)CHD2 (13)C-CEST technique can be upwards of a factor of 5 times higher than for a previously published (13)CH3 (13)C-CEST approach (Bouvignies and Kay in J Biomol NMR 53:303-310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins.
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada.
Enrico
E
Rennella
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada.
Rui
R
Huang
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada.
Algirdas
A
Velyvis
Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada. kay@pound.med.utoronto.ca.|Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. kay@pound.med.utoronto.ca.
Lewis E
LE
Kay
D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D011506:Proteins
13CHD2; CEST; Chemical exchange; Methyl-labeling; Proteasome; Sensitivity enhancement