EVOLUTIONARY ADAPTATION OF PROTEINS TO THE INTRACELLULAR ENVIRONMENT: STUDY OF THE EFFECT OF QUINARY INTERACTIONS ON THE STABILITY AND MOLECULAR DYNAMICS OF THE FF1 DOMAIN OF P190A

The cellular environment is densely crowded, and proteins undergo nonspecific weak interactions known as quinary interactions. Cellular functions rely on the competition between multiple weak and strong interactions involving protein surface forces. Since most proteins, nucleotides, and membranes are negatively charged, the intracellular environment is both crowded and polyanionic. While crowding was once thought to stabilize proteins, recent studies show that quinary interactions can either stabilize or destabilize proteins in the cytoplasm. In this work, we investigated the effects of a cytoplasmic mimetic environment using polyanionic polymers on the stability and dynamics of the FF1 domain of the guanosine triphosphatase-activating protein Rho (p190A). This domain contains four α-helices and a phosphorylation site in its hydrophobic core. Phosphorylation of the tyrosine at this site requires the protein to adopt an excited state in which two helices are unfolded. We performed 15N-1H-HSQC experiments comparing conditions with and without sodium polyacetate 8000 (NaPAc8000). Addition of NaPAc8000 caused peak line broadening and revealed residues in double conformations. While double conformations are also present in the absence of polyanions, the quinary interactions favored the minor conformational state as the predominant one. These affected residues were located both on the protein surface and within the hydrophobic core, suggesting that surface modifications induced by the polyanion propagated inward. Chemical shift perturbations were distributed across the surface, indicating no specific binding site. To assess stability (ΔGF-U), we optimized the CLEANEX NMR experiment to measure amide hydrogen exchange (kex) in water. The presence of NaPAc8000 reduced the number of solvent-exchanging residues, demonstrating increased protection from H exchange. Stability (ΔGu) was also evaluated by urea denaturation at varying NaPAc8000 concentrations through sequential 15N-1H-HSQCs, providing complementary stability measurements. Finally, we performed near-complete NMR relaxation characterization of the FF1 equilibrium with its first thermally accessible excited state. Overall, our results show that polyanions interact broadly across the protein surface, altering its conformational equilibrium and enhancing protection of the most labile residues.