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

Abstract

15 N R 2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide-water exchange can severely skew Hahn-echo based 15 N R 2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15 N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent 15 N R 2 rate constants  which should not be mistaken with protein inherent chemical exchange contributions, R ex , to 15 N R 2. For measurements of 15 N R 2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D 2 O molar fraction in the sample buffer, as low as 1%, or the use of an external D 2 O reference along with a modified 15 N R 2 Hahn-echo based experiment. This combination allows for the measurement of R ex contributions to 15 N R 2 originating from conformational exchange in a time window from µs to ms.

Structural insights into α-synuclein monomer–fibril interactions

“…N-1 H NMR experiment as described previously(47), data size 80 × 512 complex points, SW of 27.6 ( 15 N) and 16.4 ppm ( 1 H). The magnetization decay was recorded at four different relaxation decay periods (0, 50, 100, and 200 ms) in an interleaved manner with 16 scans, acquisition time ∼5 h. The 3D15 N-resolved [ 1 H, 1 H]-NOESY experiments (48) were acquired with a NOE mixing time τ m = 200 ms, 256 × 55 × 1,024 complex points, SW of 13.0 ppm ( 1 H), 35.2 ppm ( 15 N) and 14.3 ppm ( 1 H), and eight scans (acquisition time ∼2 d).…”

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain

“…Τhe errors were evaluated using the formula , ( (accessed on 14 February 2022), relax Manual [ 40 ]) where uncertainties on peak intensities, and , were estimated using two repeated points in the CPMG experiments; I 1 (ω1) and I 0 are the peak intensities at a frequency ω 1 in the CPMG series, and I 0 is the experiment performed without the constant time [ 30 ]. Only for the experiments with dilute samples, were the quantity of D2O in samples reduced to 4%, to decrease deuterium isotopic effects that affect the exchange rates [ 41 ].…”

“…As the measurements were made at 35 °C, fast hydrogen exchange occurs particularly in disordered regions of the proteins, i.e., the N- and C-termini domains. As mentioned in several publications, this may lead to R ex contributions that are associated with hydrogen exchange, but not conformational exchange [ 41 , 43 ]. These artefactual R ex exchange processes have been found to be related to deuterium isotopic effects resulting from solvent exchange occurring at timescales for which the CPMG experiments are sensitive [ 41 ].…”

“…As mentioned in several publications, this may lead to R ex contributions that are associated with hydrogen exchange, but not conformational exchange [ 41 , 43 ]. These artefactual R ex exchange processes have been found to be related to deuterium isotopic effects resulting from solvent exchange occurring at timescales for which the CPMG experiments are sensitive [ 41 ]. In our case, the N- and C-terminal residues, which are largely disordered, exchange faster with solvent, particularly at 35 °C, leading to the detection of R ex in 15 N-CPMG dispersion relaxation experiments.…”

Experimental characterization of the dynamics of IDPs and IDRs by NMR

“…The main reason is the sensitivity of the rates measured to the exchange of labile amide protons with the solvent. This process leads to apparent increase of transverse relaxation rates due to the chemical exchange between hydrogen-bound and deuterium bound nitrogen-15 14 . In addition, the starting polarization of amide proton is strongly dependent on the partial saturation of water protons, which is relaxation-delay dependent.…”