Exchangeable deuterons introduce artifacts in amide 15N CEST experiments used to study protein conformational exchange

Elucidating dissociation activation energies in host–guest assemblies featuring fast exchange dynamics

“…[9][10][11] The fact that GEST can be applied with a conventional NMR setup, which is available at any research institute, offers new opportunities to explore dissociation dynamics and E a in a variety of supramolecular systems and should provide insights into less-studied mechanisms. The extension of CEST-NMR experiments to 15 N-and 13 C-and its implementation in other dynamic molecular systems, such as proteins, 71,72 emphasizes the potential of the proposed approach to be further developed. Because state-of-the-art experimental methods for determining the rate constants of host-guest complexes were so far limited to comparably slow binding systems (<10 s À1 ), having now a method at avail that can also be applied to rapidly unbinding guests (>1000 s À1 ) may open new possibilities for shedding light on fundamental questions in host-guest complexation kinetics.…”

A Double-Resonance CEST Experiment To Study Multistate Protein Conformational Exchange: An Application to Protein Folding

“…NMR experiments were performed on a Bruker Avance III HD 700 MHz spectrometer equipped with a cold probe. Samples contained ∼1 mM [U- 15 N] T4L in 20 mM sodium phosphate, 5 mM NaN 3 , 2 mM EDTA, 2.5% d 6 -DMSO, pH 3.0 buffer, where d 6 -DMSO served as the lock 55 (see the Supporting Information for details).…”

Measuring radiofrequency fields in NMR spectroscopy using offset-dependent nutation profiles

“…The second one was used as an external standard for 15 N B 1 calibration and contained 1 mM 15 N e -labelled tryptophan prepared in the same buffer as the 15 N e -labeled tryptophan/U-15 N ubiquitin sample mentioned above. The aprotic solvent d 6 -DMSO served as the lock solvent in both samples in order to eliminate artifacts from H/D solvent exchange [47].…”

“…We chose 15 N e -labeled Trp as a suitable small molecule because of a number of favorable properties such as easy availability, the slow solvent exchange rate of the indole 1 H e [60], a sharp indole 1 H e -15 N e correlation, and a 15 N chemical shift that falls within the resonance frequency range of typical protein and nucleic acid molecules. In order to eliminate potential interference from H/D exchange in the nutation profiles, we used 2.5 % d 6 -DMSO as the lock solvent [47]. CONDENZ profiles 25Hz, 26 Hz, right) demonstrating the high sensitivity of these profiles to small changes in B 1 field amplitude.…”