Studying Intrinsically Disordered Proteins under True In Vivo Conditions by Combined Cross‐Polarization and Carbonyl‐Detection NMR Spectroscopy

Abstract

Under physiological conditions,s tudies of intrinsically disordered proteins (IDPs) by conventional NMR methods based on proton detection are severely limited by fast amide-proton exchange with water. 13 Cdetection has been proposed as as olution to the exchange problem, but is hampered by low sensitivity.W epropose anew pulse sequence combining proton-nitrogen cross-polarization and carbonyl detection to record high-resolution, high-sensitivity NMR spectra of IDPs under physiological conditions.T od emonstrate the efficacy of this approach,werecorded ahigh-quality N-CO correlation spectrum of a-synuclein in bacterial cells at 37 8 8C.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.

15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

“…68,69 and non-local and local dynamics of IDPs using mainly15 N CPMG based relaxation dispersion experiments[15][16][17][18]67,[69][70][71][72][73][74][75][76][77][78][79][80] . Several experimental strategies have been designed to allow the recording of 201 H-15 N correlation spectra81,82 and CPMG relaxation experiments of IDPs under physiological conditions and obviate the influence of amide exchange43,83 , but the adverse impact of D 2 O through the isotope effect has to our knowledge escaped attention. At physiological pH and near physiological temperatures, we observed a substantial R ex contribution induced by D 2 O in the sample buffer that is not suppressed for a low CPMG frequency of 20 kHz.…”

NMR of Macromolecular Assemblies and Machines at 1 GHz and Beyond: New Transformative Opportunities for Molecular Structural Biology

“…demonstrated a 13 C-detected NMR experiment for probing arginine side chain 15 N η/ε − 13 C ζ correlations at high magnetic field strengths, which facilitates determination of the arginine ionization states indicating conformational changes in the target protein (100). This method also makes use of J-based cross polarization, a method that has shown particular promise for disordered proteins as well (101, 102). …”

“…Enhanced sensitivity from ultrahigh magnetic fields will likely increase the efficiency of in vivo structure determination of low concentration proteins. While remarkable results have been accomplished with respect to in vivo studies of disordered proteins (179), ultrahigh fields in combination with 13 C detection methods (60, 102) will further benefit this area of research. NMR of IDPs suffers from severe sensitivity and resolution challenges due to dynamics, solvent exchange, and the lack of an ordered secondary structure.…”

“…NMR of IDPs suffers from severe sensitivity and resolution challenges due to dynamics, solvent exchange, and the lack of an ordered secondary structure. Lippens and co-workers presented a method for combined J- and CP-based correlation with 13 CO detection at 900 MHz for characterization of disordered α-synculein in E. coli cells (102). Whole cell solid-state NMR studies have covered a wide range of systems including bacteria and biofilms to address many significant questions including perturbations in bacterial cell walls induced by drug binding (180, 181), cellular composition (182) and metabolomics (183), as well as monitoring of protein expression and cellular processes (184–186).…”

Taking Simultaneous Snapshots of Intrinsically Disordered Proteins in Action

“…These are two features that have stimulated the development of exclusively heteronuclear NMR experiments based on 13 C direct detection for the study of IDPs. The two-dimensional (2D) CON experiment (15,16) is now widely used to characterize highly flexible IDPs, thanks to the excellent chemical shift dispersion of the crosspeaks observed in this experiment and the possibility to reveal atomic resolution information on IDPs also in experimental conditions in which H N resonances are not observable (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). The wide use of exclusively heteronuclear NMR experiments for protein investigations has been stimulated by improvements in instrumental sensitivity (29).…”