Abstract
Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D 1H–15N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-013-9811-1) contains supplementary material, which is available to authorized users.
Highlights
NMR spectroscopy is a versatile tool for studying macromolecular dynamics
Remarkable progress has been demonstrated in studies of equilibrium conformatinal transitions (Baldwin and Kay 2009), protein folding (Frieden et al 1993; Balbach et al 1995; Rennella et al 2012b), post-translational modifications (Selenko et al 2008; Liokatis et al 2010; Theillet et al 2012a) and other kinetic processes (Smith et al 2013)
In this work we demonstrate that 3D non-uniform sampling (NUS) BEST-TROSY HNCO (BTHNCO) experiment with co-multi-dimensional decomposition (MDD) processing can be used for quantification of the HD-exchange rates with half-times from a couple of minutes and larger
Summary
NMR spectroscopy is a versatile tool for studying macromolecular dynamics. Remarkable progress has been demonstrated in studies of equilibrium conformatinal transitions (Baldwin and Kay 2009), protein folding (Frieden et al 1993; Balbach et al 1995; Rennella et al 2012b), post-translational modifications (Selenko et al 2008; Liokatis et al 2010; Theillet et al 2012a) and other kinetic processes (Smith et al 2013). In many practical cases the duration of a 1D experiment is an order of magnitude shorter than the time of the studied reaction and the kinetics parameters can be obtained from fitting the peak intensities to an appropriate model. It is clear, that signal resolution in the 1D spectrum is not sufficient for large molecules and
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