EVOLUTIONARY ADAPTATION OF PROTEINS TO THE INTRACELULLAR ENVIRONMENT: STUDY OF THE EFFECT OF POLYANIONS ON THE STABILITY AND MOLECULAR DYNAMICS OF THE FF1 DOMAIN OF P190A

The cellular environment is a densely crowded dynamic environment where proteins are subject to non-specific weak attractive and repulsive interactions, known as quinary interactions. Specific cellular function relies on a competition between numerous weak and strong interactions, where the protein surfaces forces are involved. Since most proteins, nucleotides, and membranes carry a net-negative charge, the intracellular environment is a crowded and polyanionic. Previously, the intracellular crowding was assumed to yield a protein stabilization. Recent work has shown that the quinary interaction may also lead to destabilization of a protein in the cytoplasm. In this work, we study the effect of a cytoplasmic mimetic environment using polyanionic polymers on the stability and dynamics of the FF1 domain of Rho guanosine triphosphatase activating protein (p190A). We optimized the NMR experiment CLEANEX to quantify water exchange rates of amide hydrogens (kex) and obtain the protein stability (DG_F-U). We will show the stability of the free protein in solution and in the presence of sodium polyacetate 1200 (NaPAc1200) and 8000 (NaPAc8000). We also show the almost complete characterization, using NMR relaxation studies, of the FF1 conformational equilibrium with the first thermally accessible conformational state (excited state). We will show the effect of polyanionic NaPAc1200 and NaPAc8000 on this equilibrium.