Temperature dependence of NMR chemical shifts: Tracking and statistical analysis

Abstract

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) 1H—15N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt.

In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

“…One resonance that is the most down field overlays with that of the same proton in CP12red. Its variation with temperature follows what is expected for a residue in a disordered region [50,51]. Also, this resonance is not observed at pH 9, and this is also indicative of fast amide proton exchange with water, and therefore absence of structure [67].…”

“…The temperature dependence of the proton chemical shift is expected to be linear for a proton in a stable conformational state [51]. For a high number of amide protons in CP12ox the temperature dependence of the NMR frequency of amide proton of residues deviated from linearity, in particular those of neighboring the dynamic N-terminal region; for example the resonances assigned to residues A5, V45 as well as a high field shifted glycine resonance that is putatively assigned to the single glycine of the N-terminal region G26 (Figure S4).…”

“…Also, this resonance is not observed at pH 9, and this is also indicative of fast amide proton exchange with water, and therefore absence of structure [67]. The second resonance is always observed even at high pH, and varies with temperature with a positive slope, that is indicative of hydrogen bond (Figure 3) [50,51]. Together, these data confirm our previous SAXS analysis that suggested that the N-terminal region can be either disordered or folded [31].…”

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts

“…The temperature dependence of the proton chemical shift is expected to be linear for a proton in a stable conformational state [ 51 ]. For a high number of amide protons in CP12 ox , the temperature dependence of the NMR frequency of amide protons of residues deviated from linearity, in particular those neighboring the dynamic N -terminal region; for example, the resonances assigned to residues A 5 , V 45 , as well as a high field-shifted glycine resonance that is putatively assigned to the single glycine of the N -terminal region G 26 ( Figure S4 ).…”

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing

“…Amide proton resonances are predominantly influenced by their hydrogen-bonded state, and the temperature-dependent changes are predominantly linear, reflecting protein thermal expansion and a corresponding increase in hydrogen bond length 50 , 86 , 87 . Small non-linear deviations of the temperature dependence of amide proton chemical shifts are sometimes detected 49 , 88 . These curved profiles result from accessible conformational exchange between ground and excited protein states 50 .…”