The Denaturant- and Mutation-Induced Disassembly of Pseudomonas aeruginosa Hexameric Hfq Y55W Mutant |
| |
Authors: | Victor Marchenkov Natalia Lekontseva Natalia Marchenko Ivan Kashparov Victoriia Murina Alexey Nikulin Vladimir Filimonov Gennady Semisotnov |
| |
Affiliation: | Institute of Protein Research, Russian Academy of Sciences, Institutskaya Street 4, 142290 Pushchino, Russia; (V.M.); (N.L.); (N.M.); (I.K.); (V.M.); (A.N.) |
| |
Abstract: | Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower. |
| |
Keywords: | Hfq hexamer mutations unfolding intermediates fluorescence thermodynamics |
|
|