**5. Conclusions**

While pressure perturbation allows one to finely and reversibly tune the stability of a protein and to modulate the rate of a conformational exchange, NMR spectroscopy can bring the spatial and temporal resolution necessary for the description of the protein folding energy landscape. Thus, HP-NMR, combining pressure perturbation, and NMR spectroscopy can give, at a residue-level resolution, an accurate structural and dynamical description of the protein folding energy landscape, revealing the existence of intermediate states as well as the rates of the associated local rearrangements.

Beyond this fundamental interest, a better understanding of protein folding/unfolding mechanisms is mandatory in many fields. Protein misfolding is involved in most of the neurodegenerative diseases, such as Alzheimer, Parkinson, prion disease, etc. The comprehensive study of the folding mechanism of the proteins specifically involved in these diseases [75–78] might allow the rational design of more efficient drugs [79]. Giving clues on the phenomena underlying protein stability, such studies can also be meaningful for the design of industrial enzymes able to work at high pressure. Thus, understanding how a protein can accommodate mutations to gain stability, keeping its function intact, has an important economic impact. Combining NMR with high pressure is an extremely powerful approach in this particular field, providing rigorous answers to important questions.

**Author Contributions:** C.D. and I.H. prepared the protein samples and carried out HP-NMR experiments. P.B. made the NMR resonance assignment of Titin I27 domain and wrote MATLAB software for the analysis of HP-NMR experiments. C.R. supervises and coordinates the projects in the HP-NMR group. He conceived experiments and analysis, participated in the interpretation, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by French Infrastructure for Integrated Structural Biology (FRISBI) grant No. ANR-10-INSB-05.

**Conflicts of Interest:** The authors declare no conflict of interest.
