Abstract
NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions.
Highlights
NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes
Solution-state NMR spectroscopy is a powerful tool for the label-free characterization of structural and dynamical aspects of biomolecular interactions and equilibria[1,2]
NMR lineshape analysis, referred to as dynamic NMR, is a well-established method for the quantitative analysis of titration data based upon the fitting of one-dimensional spectra to theoretical or numerical solutions of the equations governing evolution of magnetization in an exchanging system[7,8,9]
Summary
NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. Following changes in two-dimensional NMR spectra of macromolecules upon titration of a binding partner is a very common and information-rich approach capable of simultaneously characterizing thermodynamic (dissociation constant), kinetic (association and dissociation rates) and structural (chemical shift) aspects of interactions[3].
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