**4. "Local" Thermodynamic and Kinetic Parameters for Folding**/**Unfolding Reactions Obtained from 2D High-Pressure NMR Spectroscopy**

In most of the studies reported in the literature, the folding/unfolding reaction of a protein is approximated by a two-state model, excluding the existence of folding intermediates in its folding energy landscape. This is in fact a rough approximation: following the more appropriate model of foldons [52,53], most globular proteins should deviate from a two-state folding mechanism by populating folding intermediates. This apparent discrepancy comes from the very low population of the intermediate states at equilibrium, due to their low stability, which hampers their detection by the usual spectroscopic methods. In addition, spectroscopies often focus on only one observable (intrinsic fluorescence of a tryptophan residue, methyl NMR resonance, etc.), yielding values for the thermodynamic parameters that are supposed to reflect the global stability. We have seen previously that these values are also affected by the local stability of the protein: in the case of Titin I27, significantly different values were obtained for ∆*V* <sup>0</sup> and ∆*G* 0 from the pressure dependence of the resonances corresponding to either the unfolded state methyl groups or to the indole NH of the tryptophan side chain. Thus, a better description of the protein folding energy landscape, including the identification of folding intermediates, can be obtained by a multiple probes analysis of the folding process.

Multidimensional homo- or heteronuclear NMR spectroscopy provides an intrinsic multi-probe approach yielding residue specific information, through correlation spectroscopy involving nuclei located on the peptide backbone (1H and <sup>15</sup>N of amide groups, <sup>1</sup>Hα and <sup>13</sup>Cα). Amide protons offer ideal probes to monitor the unfolding reaction: each amino acid bears an NH group, with the exception of proline, generally a minority residue in the composition of soluble proteins (< 3%), and will give rise to a specific correlation in the usually well-resolved 2D [1H-15N] HSQC spectrum of the native protein. In addition, proton/deuteron exchange measurement for amide protons has been extensively used to evaluate the local stability of a protein [54], bringing important information on local unfolding phenomena.
