Enhanced accuracy of kinetic information from CT-CPMG experiments by transverse rotating-frame spectroscopy.
Micro-to-millisecond motions of proteins transmit pivotal signals for protein function. A powerful technique for the measurement of these motions is nuclear magnetic resonance spectroscopy. One of the most widely used methodologies for this purpose is the constant-time Carr-Purcell-Meiboom-Gill (CT-CPMG) relaxation dispersion experiment where kinetic and structural information can be obtained at atomic resolution. Extraction of accurate kinetics determined from CT-CPMG data requires refocusing frequencies that are much larger than the nuclei's exchange rate between states. We investigated the effect when fast processes are probed by CT-CPMG experiments via simulation and show that if the intrinsic relaxation rate (R(CT-CPMG)(2,0)) is not known a priori the extraction of accurate kinetics is hindered. Errors on the order of 50 % in the exchange rate are attained when processes become fast, but are minimized to 5 % with a priori (CT-CPMG)(2,0)) information. To alleviate this shortcoming, we developed an experimental scheme probing (CT-CPMG)(2,0)) with large amplitude spin-lock fields, which specifically contains the intrinsic proton longitudinal Eigenrelaxation rate. Our approach was validated with ubiquitin and the Oscillatoria agardhii agglutinin (OAA). For OAA, an underestimation of 66 % in the kinetic rates was observed if (CT-CPMG)(2,0)) is not included during the analysis of CT-CPMG data and result in incorrect kinetics and imprecise amplitude information. This was overcome by combining CT-CPMG with (CT-CPMG)(2,0)) measured with a high power R1ρ experiment. In addition, the measurement of (CT-CPMG)(2,0)) removes the ambiguities in choosing between different models that describe CT-CPMG data.
Department for NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany.
David
D
Ban
Adam
A
Mazur
Marta G
MG
Carneiro
T Michael
TM
Sabo
Karin
K
Giller
Leonardus M I
LM
Koharudin
Stefan
S
Becker
Angela M
AM
Gronenborn
Christian
C
Griesinger
Donghan
D
Lee
D001426:Bacterial Proteins / Q000737:chemistry*; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D054300:Oscillatoria / Q000737:chemistry; D011487:Protein Conformation; D025801:Ubiquitin / Q000737:chemistry*
D001426:Bacterial Proteins; D037102:Lectins; C415125:OAA protein, Oscillatoria agardhii; D025801:Ubiquitin