Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR

SIMPLE SUMMARY: During the last decade, high-pressure multidimensional NMR has emerged as a very powerful tool to describe the folding landscapes of proteins. This is (i) because pressure is a gentle perturbation, the effects of which originate from local properties of the folded state, contrary to chemical or thermal denaturation, and (ii) because multidimensional NMR intrinsically provides multiple probes strategically scattered on the three-dimensional structure of the protein, allowing a quasi-atomic resolution to describe the folding pathway. Residue-specific information obtained from these probes can be used to describe protein folding pathways through the calculation of NMR-derived fractional probabilities of contact at increasing pressure. Here, we used this strategy to evaluate and compare the results obtained from NH amide, CαHα, and CH(3) groups when used as NMR probes to explore the folding pathway of the model protein ∆+PHS Staphylococcal Nuclease. ABSTRACT: Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CαHα groups are strategically located on the backbone of the protein, while CH(3) groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CαHα, and CH(3) groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CαHα when compared to amide groups.