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.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…Even though less sensitive than 1 H– 15 N HSQC experiments, these 13 C-detection experiments provided much improved spectral dispersion. Moreover, the 13 Cα 13 CO experiments can produce spectra of IDPs in all conditions of pH and temperature, which is not always the case for 13 CO 15 N spectra, and not the case at all for 1 H– 15 N schemes, because of the water-amide proton exchange . Recent 13 Cα 13 CO pulse sequences were proposed, which can produce exploitable 2D spectra of proteins at ∼20 μM in ∼1 h. , 13 CO-direct detection approaches are however not likely to be efficient for globular proteins in cells, because of the large chemical shift anisotropy of 13 CO resonances in peptides.…”

“…To properly quantify protein populations in states A and B based on their respective NMR peak intensities, it is important to also quantify the relaxation properties of the exploited peaks, or at least to know the intrinsic intensities of the pure states A or B. Danielsson and Oliveberg provided an impeccable example in their study of the folded/unfolded populations of SOD1 barrel‑I35A . Great variations of amide 1 H N -NMR signal intensities are also observed for disordered regions of proteins upon pH or temperature changes, due to water:amide H-exchange. ,,− It is thus advisable to carry out a careful characterization of the intracellular pH and of the signal intensity per molecule for every residue.…”

“…358 Great variations of amide 1 HN-NMR signal intensities are also observed for disordered regions of proteins upon pH or temperature changes, due to water:amide proton exchange. 428,436,[529][530][531] It is thus advisable to carry out a careful characterization of the intracellular pH and of the signal intensity per molecule for every residue.…”

“…Improved pulse sequences and 13 C-optimized probes give access to exploitable NMR spectra of disordered proteins at ~25 µM in about one hour at 37 ºC. 436 Whether they would yield such performances in cells remains to be tested.…”

“…111,438 While in situ production can accommodate a number of isotope-labeling schemes in E. coli, there are a number of labeling possibilities to explore in insect and mammalian cells. These might also permit to use 13 C-direct detection, which appears to be necessary to characterize disordered proteins at physiological temperatures, 178,436 together with and 1 Ha- 13 Ca correlations. [932][933][934] Low-gamma nuclei detection may actually be adapted to the slower tumbling times observed in cells, notably in absence of uniform deuteration.…”