Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment

Abstract

Protein backbone 15N spin relaxation rates measured by solution NMR provide useful dynamic information with a site-specific resolution. The conventional method is to record a series of 2D 1H- 15N HSQC spectra with varied relaxation delays, and derive relaxation rate from the following curve fitting on the resonance intensities. Proteins with poorly resolved spectra often require several 3D HNCO spectra to be collected on a 15N/ 13C double labeled protein sample. In order to reduce the relaxation dimension Carr et al . (P.A. Carr, D.A. Fearing, A.G. Palmer, 3D accordion spectroscopy for measuring N-15 and (CO)-Carbon-13 relaxation rates in poorly resolved NMR spectra, J. Magn. Reson. 132 (1998) 25–33) employed an Accordion type HNCO pulse sequence to obtain 15N or 13C T 1 relaxation rates by numerical fitting of the relaxation interfered free induction decay (FID) data. To avoid intensive analysis of the time domain data, we propose a modified protocol to measure 15N T 1 and T 2 relaxation rates from easily obtained line-widths in an Accordion HNCO spectrum. Both T 1 and T 2 relaxation could be simultaneously convoluted into the constant-time evolution periods of 13C′ and 15N, respectively. The relaxation delay was allowed to reach at least 3 × T 1 or 3 × T 2 so that the signal was substantially decayed by the end of the FID, and the resulting peak full-width at half height (FWHH) could be directly used to calculate relaxation rate. When applied to the 76-residue Ubiquitin and the 226-residue glutamine-binding protein (GlnBP), this method yielded T 1 and T 2 values deviating on average by 4–6% and 5–7%, respectively, from the measurements based on the conventional 2D method. In comparison, the conventional methods possessed intrinsic error ranges of 2–4% for T 1 and 3–6% for T 2. In addition to comparable accuracy, the fully-relaxed Accordion HNCO method presented here allowed measurements of relaxation rates for resonances unresolved in 2D spectra, thus providing a more complete dynamic picture of the protein.