Recent Advances in Solution NMR: Fast Methods and Heteronuclear Direct Detection

Abstract

Today, NMR spectroscopy is the technique of choice to investigate molecular structure, dynamics, and interactions in solution at atomic resolution. A major limitation of NMR spectroscopy for the study of biological macromolecules such as proteins, nucleic acids, and their complexes, has always been its low sensitivity, a consequence of the weak magnetic spin interactions. Therefore many efforts have been invested in the last decade to improve NMR instrumentation in terms of experimental sensitivity. As a result of these efforts, the availability of high-field magnets, cryogenically cooled probes, and probably in the near future hyperpolarization techniques, the intrinsic NMR sensitivity has increased by at least one order of magnitude. Stimulated by new challenges in the life sciences, these technical improvements have triggered the development of new NMR methods for the study of molecular systems of increasing size and complexity. Herein, we focus on two examples of recently developed NMR methodologies. First, advanced multidimensional data acquisition schemes provide a speed increase of several orders of magnitude. Second, NMR methods based on the direct detection of low-gamma nuclei present a new spectroscopic tool, highly complementary to conventional NMR techniques. These new methods provide powerful new NMR tools for the study of short-lived molecules, large and intrinsically unstructured proteins, paramagnetic systems, as well as for the characterization of molecular kinetic processes at atomic resolution. These examples illustrate how NMR is continuously adapting to the new challenges in the life sciences, with the focus shifting from the characterization of single biomolecules to an integrated view of interacting molecular networks observed at varying levels of biological organization.

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

“…[28,32] Indeed carbonyl carbon and nitrogen are among the nuclei that retain the largest chemical-shift dispersion also for systems that lack a stable three-dimensional structure, and the inter-residue nature of the C'ÀN correlation contributes to increasing the chemical-shift dispersion. [90] Proline residues and X-Pro peptide bonds, which are a characteristic feature of highly flexible segments and thus are very abundant in flexible linkers, as well as in intrinsically disordered proteins, can be extensively characterized through the experiments proposed here. Finally, the proposed experiments, if converted to 13 C-start rather than 1 Hstart versions, constitute a valuable tool to characterize paramagnetic proteins, by providing information also in regions of the protein where 1 H resonances are too broad to be detected.…”

A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins

“…For example, a combination of five-, six-and sevendimensional HN-detected experiments, which were recorded using automated projection spectroscopy (APSY; Hiller et al 2005) in combination with the assignment software MARS (Jung and Zweckstetter 2004), allowed automatic assignment of 92 % of the assignable residues of the 441-residue IDP Tau (Narayanan et al 2010). In addition, 13 C-detection is a very powerful approach to increase signal dispersion and reduce line broadening, which is either caused by solvent exchange or conformational exchange (Felli and Brutscher 2009;Skora et al 2010). 13 C-detected NMR experiments (Bermel et al 2006a(Bermel et al , b, 2012aBertini et al 2004;Pervushin and Eletsky 2003;Shimba et al 2004;Takeuchi et al 2010), in particular when combined with non-uniform sampling techniques (Barna et al 1987;Bermel et al 2012bBermel et al , 2013Chylla and Markley 1995;Coggins and Zhou 2006;Holland et al 2011;Kazimierczuk et al 2006;Kazimierczuk and Orekhov 2011;Korzhneva et al 2001;Novacek et al 2011;Orekhov et al 2001), can therefore enable the resonance assignment and structural characterization of large IDPs (Csizmok et al 2008;Narayanan et al 2010;Novacek et al 2013;Zawadzka-Kazimierczuk et al 2012a).…”

Recent progress in NMR spectroscopy: Toward the study of intrinsically disordered proteins of increasing size and complexity

“…The large heteronuclear chemical shift dispersion makes exclusively heteronuclear experiments particularly well suited for the exploitation of reduced or sparse sampling methods in the indirect dimensions (32, 36). These approaches combined with the LRE enable acquisition of multidimensional experiments with each additional dimension providing an increase in cross peak dispersion and information content (45). All these features (Fig.…”