Divided-evolution-based pulse scheme for quantifying exchange processes in proteins: powerful complement to relaxation dispersion experiments.
A method for quantifying millisecond time scale exchange in proteins is presented based on scaling the rate of chemical exchange using a 2D (15)N, (1)H(N) experiment in which (15)N dwell times are separated by short spin-echo pulse trains. Unlike the popular Carr-Purcell-Meiboom-Gill (CPMG) experiment where the effects of a radio frequency field on measured transverse relaxation rates are quantified, the new approach measures peak positions in spectra that shift as the effective exchange time regime is varied. The utility of the method is established through an analysis of data recorded on an exchanging protein-ligand system for which the exchange parameters have been accurately determined using alternative approaches. Computations establish that a combined analysis of CPMG and peak shift profiles extends the time scale that can be studied to include exchanging systems with highly skewed populations and exchange rates as slow as 20 s(-1).
Departments of Molecular Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Guillaume
G
Bouvignies
D Flemming
DF
Hansen
Pramodh
P
Vallurupalli
Lewis E
LE
Kay
D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013997:Time Factors
D029701:Saccharomyces cerevisiae Proteins; C491504:ARK1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases