title: Proton-decoupled CPMG: a better experiment for measuring (15)N R2 relaxation in disordered proteins.
abstract: (15)N R2 relaxation is one of the most informative experiments for characterization of intrinsically disordered proteins (IDPs). Small changes in nitrogen R2 rates are often used to determine how IDPs respond to various biologically relevant perturbations such as point mutations, posttranslational modifications and weak ligand interactions. However collecting high-quality (15)N relaxation data can be difficult. Of necessity, the samples of IDPs are often prepared with low protein concentration and the measurement time can be limited because of rapid sample degradation. Furthermore, due to hardware limitations standard experiments such as (15)N spin-lock and CPMG can sample the relaxation decay only to ca. 150ms. This is much shorter than (15)N T2 times in disordered proteins at or near physiological temperature. As a result, the sampling of relaxation decay profiles in these experiments is suboptimal, which further lowers the precision of the measurements. Here we report a new implementation of the proton-decoupled (PD) CPMG experiment which allows one to sample (15)N R2 relaxation decay up to ca. 0.5-1s. The new experiment has been validated through comparison with the well-established spin-lock measurement. Using dilute samples of denatured ubiquitin, we have demonstrated that PD-CPMG produces up to 3-fold improvement in the precision of the data. It is expected that for intrinsically disordered proteins the gains may be even more substantial. We have also shown that this sequence has a number of favorable properties: (i) the spectra are recorded with narrow linewidth in nitrogen dimension; (ii) (15)N offset correction is small and easy to calculate; (iii) the experiment is immune to various spurious effects arising from solvent exchange; (iv) the results are stable with respect to pulse miscalibration and rf field inhomogeneity; (v) with minimal change, the pulse sequence can also be used to measure R2 relaxation of (15)N(ε) spins in arginine side chains. We anticipate that the new experiment will be a valuable addition to the NMR toolbox for studies of IDPs.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Tairan
authors.initials: T
authors.lastname: Yuwen
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA. Electronic address: nikolai@purdue.edu.
authors.forename: Nikolai R
authors.initials: NR
authors.lastname: Skrynnikov
mesh_terms: D000465:Algorithms; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002138:Calibration; D002645:Chickens; D003198:Computer Simulation; D006801:Humans; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D013049:Spectrin / Q000737:chemistry; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D025801:Ubiquitin; D013049:Spectrin; D001120:Arginine
keywords: (15)N relaxation; Arginine side-chain dynamics; Backbone dynamics; CPMG experiment; Intrinsically disordered proteins; Solvent exchange; Spin simulations; Spin-lock experiment; Ubiquitin; α-Spectrin SH3 domain