Hfq from Pseudomonas aeruginosa is a posttranslational regulator of gene expression that binds small noncoding RNAs (sRNA) and promotes their interaction with mRNAs. It belongs to the widespread Sm/Lsm (Sm-like) protein family and forms hexamers in crystals and solution. In spite of the mesophilic source Hfq possess extremely high thermostability. Measurements of the protein stability by microcalorimetry showed the transition midpoint for Hfq was about 120°C at neutral pH. A model for a temperature-induced unfolding of the Hfq hexamers which includes dissociation to monomers and their subsequent unfolding was suggested. To confirm this hypothesis it was necessary to obtain Hfq monomer by complex substitution of amino acids Asp9, Val43 and Tyr55, which organized inter-monomer hydrogen bonds in the protein hexamer. The obtained mutant form of the Hfq was studied by semi-native electrophoresis, size-exclusion chromatography and dynamic light scattering.
Hfq, Sm-like proteins, oligomerization, semi-native electrophoresis, dynamic light scattering
1. Shapiro L., Franze de Fernandez M.T., August J.T. Resolution of two factors required in the Q-beta-RNA polymerase reaction. Nature, 1968, vol. 220, pp. 478-480.
2. de Haseth P.L., Uhlenbeck O.C. Interaction of Escherichia coli host factor protein with Q beta ribonucleic acid. Biochemistry, 1980, vol. 19, no. 26, pp. 6146-51.
3. Valentin-Hansen P., Eriksen M. The bacterial Sm-like protein Hfq : a key player in RNA transactions. Mol. Microbiol., 2004, vol. 51, pp. 1525-1533.
4. Achsel T., Stark H., Lührmann R. The Sm domain is an ancient RNA-binding motif with oligo(U) specificity. Proc. Natl. Acad. Sci. U. S. A., 2001, vol. 98, pp. 3685-3689.
5. Shikama N., Lutz W., Kretzschmar R., Sauter N., Roth J.F., Marino S., Wittwer J., Scheidweiler A., Eckner R. Essential function of p300 acetyltransferase activity in heart, lung and small intestine formation. EMBO J., 2003, vol. 22, pp. 5175-5185.
6. Murina V.N., Melnik B.S., Filimonov V.V., Ühlein M., Weiss M.S., Müller U., Nikulin A.D. Effect of conserved intersubunit amino acid substitutions on Hfq protein structure and stability. Biochem., 2014, vol. 79, pp. 469-477.
7. Nikulin A., Stolboushkina E., Perederina A., Vassilieva I., Blaesi U., Moll I., Kachalova G., Yokoyama S., Vassylyev D., Garber M. [et al.] Structure of Pseudomonas aeruginosa Hfq protein. Acta Crystallogr. D. Biol. Crystallogr., 2005, vol. 61, pp. 141-146.
8. Panja S., Woodson S. Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing. J. Mol. Biol., 2012, vol. 417, pp. 406-412.
