Temperature-dependence of protein hydrogen bond properties as studied by high-resolution NMR

The temperature-dependence of a large number of NMR parameters describing hydrogen bond properties in the protein ubiquitin was followed over a range from 5 to 65°C. The parameters comprise hydrogen bond (H-bond) scalar couplings, h3JNC′, chemical shifts, amide proton exchange rates, 15N relaxation parameters as well as covalent 1JNC′ and 1JNH couplings. A global weakening of the h3JNC′ coupling with increasing temperature is accompanied by a global upfield shift of the amide protons and a decrease of the sequential 1JNC′ couplings. If interpreted as a linear increase of the N⋯O distance, the change in h3JNC′ corresponds to an average linear thermal expansion coefficient for the NH→O hydrogen bonds of 1.7×10−4/K, which is in good agreement with overall volume expansion coefficients observed for proteins. A residue-specific analysis reveals that not all hydrogen bonds are affected to the same extent by the thermal expansion. The end of β-sheet β1/β5 at hydrogen bond E64→Q2 appears as the most thermolabile, whereas the adjacent hydrogen bond I3→L15 connecting β-strands β1 and β2 is even stabilized slightly at higher temperatures. Additional evidence for the stabilization of the β1/β2 β-hairpin at higher temperatures is found in reduced hydrogen exchange rates for strand end residue V17. This reduction corresponds to a stabilizing change in free energy of 9.7 kJ/mol for the β1/β2 hairpin. The result can be linked to the finding that the β1/β2 hairpin behaves as an autonomously folding unit in the A-state of ubiquitin under changed solvent conditions. For several amide groups the temperature-dependencies of the amide exchange rates and H-bond scalar couplings are uncorrelated. Therefore, amide exchange rates are not a sole function of the hydrogen bond “strength” as given by the electronic overlap of donors and acceptors, but are clearly dependent on other blocking mechanisms.