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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 118; no. 34; pp. 1 - 9
• Main Authors: Okuno, Yusuke, Yoo, Janghyun, Schwieters, Charles D., Best, Robert B., Chung, Hoi Sung, Clore, G. Marius
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 24.08.2021
• Subjects: Animals; Biological Sciences; Biopolymer denaturation; Destabilization; Drosophila melanogaster; Drosophila Proteins - chemistry; Drosophila Proteins - metabolism; Electrostatic properties; Equilibrium; Models, Molecular; NMR; Nuclear magnetic resonance; Physical Sciences; Protein Denaturation; Protein Folding; Protein interaction; Protein Unfolding; Proteins; Solvents; Solvents - chemistry; src Homology Domains; Thermodynamics; drkN SH3 native and unfolded states; protein–cosolute interactions; transient states; NMR relaxation; replica exchange molecular dynamics
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2112021118

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.