Quantifying millisecond exchange dynamics in proteins by CPMG relaxation dispersion NMR using side-chain 1H probes.
A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the β positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the γ positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstrated with an application to the four-helix bundle colicin E7 immunity protein, Im7, which folds via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. The extracted (1)H chemical shift differences at side-chain positions provide valuable restraints in structural studies of invisible, excited states, complementing backbone chemical shifts that are available from existing relaxation dispersion experiments.
Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Alexandar L
AL
Hansen
Patrik
P
Lundström
Algirdas
A
Velyvis
Lewis E
LE
Kay
D000596:Amino Acids / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002621:Chemistry / Q000379:methods; D003087:Colicins / Q000737:chemistry; D017666:Deuterium Oxide / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D007700:Kinetics; D008279:Magnetic Resonance Imaging / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D012997:Solvents / Q000737:chemistry; D013997:Time Factors
D000596:Amino Acids; D002352:Carrier Proteins; D003087:Colicins; C509253:E colicin-binding immunity protein Im7, E coli; D029968:Escherichia coli Proteins; D011506:Proteins; D011522:Protons; D012997:Solvents; D005947:Glucose; D017666:Deuterium Oxide