Atomic view of cosolute-induced protein denaturation probed by NMR solvent paramagnetic relaxation enhancement

Abstract

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute–protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute–protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, 〈r−6〉norm, and an effective correlation time, τc. The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute–protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the 〈r−6〉norm values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

Theory and Applications of Nitroxide-based Paramagnetic Cosolutes for Probing Intermolecular and Electrostatic Interactions on Protein Surfaces

“…Solvent paramagnetic relaxation enhancement (sPRE) has been used to study protein solvent accessibility, 1−16 to refine NMR protein structures, 17−19 to investigate protein−cosolute interactions, 20,21 and to characterize the electrostatic potential of small molecules and proteins. 22−29 The paramagnetic moiety of the cosolutes employed in these studies comprises either a paramagnetic metal ion or a nitroxide free radical.…”

Reframing the Protein Folding Problem: Entropy as Organizer

“…Unlike our model, a recent mechanistic proposal for osmolyte denaturation reported direct binding between the cosolvent and the backbone using 3-carbamoyl-PROXYL and 3-carboxy-PROXYL as cosolvents in lieu of a naturally occurring osmolyte . Many cosolvents can denature proteins but not necessarily by similar mechanisms.…”

“…56 Unlike our model, a recent mechanistic proposal for osmolyte denaturation reported direct binding between the cosolvent and the backbone using 3-carbamoyl-PROXYL and 3-carboxy-PROXYL as cosolvents in lieu of a naturally occurring osmolyte. 57 Many cosolvents can denature proteins but not necessarily by similar mechanisms. For example, urea (an organic osmolyte) and guanidine hydrochloride (a salt) are both strong denaturants, but with entirely different molecular mechanisms, although both interact preferentially with the unfolded state.…”

Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions

“…The paramagnetic group attenuates resonance intensities of residues by enhancing relaxation in a distance-dependent manner (46). PRE is suitable for probing transient interactions, including the weak interactions between co-solutes and macromolecules (46,47) and the intermolecular interactions that proceed phase separation (48,49). A higher PRE value indicates that peptides come closer to the residue analyzed.…”