Vol.:(0123456789) 1 3 Journal of Biomolecular NMR https://doi.org/10.1007/s10858-018-00223-3 ARTICLE Exchangeable deuterons introduce artifacts in amide 15 N CEST experiments used to study protein conformational exchange Ved Prakash Tiwari 1  · Subhendu Pandit 1  · Pramodh Vallurupalli 1 Received: 14 November 2018 / Accepted: 27 December 2018 © Springer Nature B.V. 2019 Abstract Protein molecules sample diferent conformations in solution and characterizing these conformations is crucial to under- standing protein function. 15 N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a ‘visible’ major state and ‘invisible’ minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope efect on the amide 15 N chemical shifts. However at moderately high temperatures (~ 50 °C) that are sometimes required to populate protein minor conform- ers to levels (~ 1%) that can be detected by CEST experiments solvent H/D exchange can lead to ‘dips’ in low B 1 15 N CEST profles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~ 18 kDa T4 Lysozyme (T4L) at 50 °C and the ~ 11 kDa E. coli hibernation promoting factor (HPF) at 52 °C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D 2 O lock or by using a small amount (~ 1–3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer. Keywords Chemical exchange · 15 N CEST · H/D exchange · Protein dynamics Introduction Protein molecules interconvert between diferent conforma- tional states in solution and understanding the conforma- tional dynamics and obtaining structures of the various con- formers is necessary to understand protein function (Austin et al. 1975; Karplus 2000; Palmer 2014; Sekhar and Kay 2013). Often exchange is between a dominant major state conformer and minor state conformer(s). Resonances arising from the minor states are invisible in traditional NMR spec- tra as the minor states have low populations to begin with and due to their short lifetimes, the resonances arising from the minor states will have large linewidths (Cavanagh et al. 2006). Over the last several years diferent NMR methods have been developed to detect the minor states by studying the relaxation properties of the visible major state peaks. R 1ρ (Palmer and Massi 2006) and CPMG type relaxation dispersion experiments (Palmer et al. 2001; Sekhar and Kay 2013) are used to detect minor states with µs to millisecond lifetimes while CEST experiments are now routinely used to detect minor states with lifetimes between 5 and 50 ms that are in slow exchange with the major state (Vallurupalli et al. 2017). In a typical CEST experiment a weak B 1 feld (5–50 Hz) is applied for a time T EX (~ 200–600 ms) and its efect on the visible major peak resonance is monitored as a function of the frequency at which the B 1 feld is applied (Forsen and Hofman 1963; Ward et al. 2000). The initial magnetization is along the Z axis and the magnetization that is along the Z axis at the end of the T EX period is detected. Dips in the intensity (I) versus frequency ν CEST (Hz) profles are observed at the position of major and minor states that are in exchange with the major state. The exchange rate(s), minor state population(s) and chemical shift(s) of the minor states are readily obtained from the analysis of the intensity profles. CEST experiments have been developed to study exchange at several sites in proteins (Bouvignies et al. 2014; Fawzi et al. 2011; Hansen et al. 2013; Vallurupalli et al. 2012; Yuwen et al. 2017) and RNA (Zhao et al. 2014) and have been used to study various process like protein folding * Pramodh Vallurupalli pramodh@tifrh.res.in 1 TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/P, Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad, Telangana 500107, India