Sensitivity‐Enhanced ¹³C‐NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH

Abstract

Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue‐specific level using 1H‐15N NMR spectroscopy, but is often limited by low signal‐to‐noise at pH>7 and T>293 K. We have developed an improved 13Cα‐13CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic‐resolution information in physiological conditions down to 25 μm. We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.

Monitoring the Interaction of α‐Synuclein with Calcium Ions through Exclusively Heteronuclear Nuclear Magnetic Resonance Experiments

“…Here we would like to present a novel set of 2D NMR experiments to follow how the properties of IDPs/IDRs change in different, physiologically relevant, experimental conditions. Based on carbonyl carbon direct detection, these experiments provide information on backbone and side‐chain chemical shifts as well as on the impact of solvent exchange at the residue level, even for those residues whose amide proton is not directly detectable. This set of 2D exclusively heteronuclear NMR experiments is tested on α‐synuclein and then used to focus on its interaction with Ca 2+ , a potential trigger for the onset of Parkinson's disease.…”

On‐Cell NMR Contributions to Membrane Receptor Binding Characterization

“…[6,[42][43][44] Therefore, SS-NMR employing heteronuclear 13 C/ 15 N-detected HR-MAS (high-resolution magic angle spinning) NMR experiments is a very promising approach. [45][46][47] Using this technique, detailed information about large structures on living cells, for example, keratin, collagen and other components of the ECM, were achieved. [48][49][50][51][52] Another way of increasing the sensitivity in SS-NMR is by exploiting the enhancement of dynamic nuclear polarization (DNP) by the usage of paramagnetic labels.…”

“…Fortunately, signal intensities in SS‐NMR are insensitive to the molecular rotational tumbling rate and thus allows to study non‐soluble or large cell surface macromolecules [6,42–44] . Therefore, SS‐NMR employing heteronuclear 13 C/ 15 N‐detected HR‐MAS (high‐resolution magic angle spinning) NMR experiments is a very promising approach [45–47] . Using this technique, detailed information about large structures on living cells, for example, keratin, collagen and other components of the ECM, were achieved [48–52] .…”

Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions

“…All these properties make the new SHACA-HSQC attractive to be used as an essential reference, which is ready for incorporation into 3D-type sequences to provide a complete set of triple-resonance experiments for uncompromised studies of IDPs. It will allow us to address special structural questions, for example, the residue-specific identification of post-translational modifications, with utmost spectral resolution and sensitivity. Furthermore, the generalized approach of the presented band-selective pure shift spectra can be of use in all types of chemical, biological, or pharmaceutical applications involving molecules with high abundance of NMR-active isotopes, including reaction monitoring, the characterization of compound libraries, or the determination of metabolic pathways. − …”