*3.3. Wide-Line NMR*

We have reviewed the varieties of radiofrequency excitations applied in wide-line NMR in two book chapters [23,24], and here we only address the simplest excitation protocol that uses a 90◦ (π/2) radiofrequency pulse. NMR measurements and data acquisition were performed with a Bruker AVANCE III NMR pulse spectrometer at a frequency of 82.4 MHz with a stability of better than 10−6. The π/2 pulse was 3–4 μs, and the dead time of the spectrometer was 6–8 μs. The inhomogeneity of the magnetic field was 2 ppm. The accumulated repeat number of the measurements was between 50 and 80.

The temperature was controlled by an open-cycle Janis cryostat with an uncertainty better than 0.5 K. The system was complemented by an adequate NMR head and by a closed sample holder.

#### **4. Conclusions**

By reinterpreting our previous results, we have determined the energy distribution of the potential barriers inhibiting the movement of water molecules bound to two protein molecules in aqueous solution. Based on our results, we could deduce quantitative conclusions about the ratios of the globular/ordered and more solvent exposed/disordered regions of the protein molecules and the extent of the latter, as well as the energy relations of the protein–water bonds. We suggest that short range forces (H-bonds) play a dominant role in the formation of the first hydrate shell.

The mapping of the water-binding characteristics of protein molecules is certainly not the only area of the application of wide-line NMR measurements and this novel interpretational procedure. The rapid, non-disruptive measurement and the data interpretation had already opened a novel avenue to study molecular interactions and to determine the moisture content of solid phase samples. In the outline of our previous work [9], we listed some additional possibilities. Three of these are also mentioned here: (i) the possibility to directly demonstrate the interaction between different molecules (e.g., protein and drug), (ii) the possibility of direct, non-destructive measurement of the different bonds between identical molecules, and (iii) the possibility to determine the effect of a standard (often NaCl containing) solvent on the structure and properties of protein molecules.

**Author Contributions:** Conceptualization, K.T., M.B., and P.T.; Methodology, M.B.; Validation, K.T., M.B. and P.T.; Formal Analysis, M.B.; Investigation K.T. and M.B.; Data Curation, M.B.; Writing-Original Draft, K.T. Writing—Review & Editing K.T., M.B. and P.T.; Visualization M.B.; Supervision, P.T. Funding Acquisition P.T.

**Funding:** This research was funded by the Hungarian Scientific Research Fund (OTKA, grant no. K124670).

**Acknowledgments:** We would like to thank all colleagues, who contributed to previous studies that resulted in the results on which this work is based.

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

#### **Abbreviations**

