Apomyoglobin is a convenient model for in vitro studies of folding/unfolding of globular proteins. Herein we describe the results of kinetic studies of folding/unfolding of mutant forms of apomyoglobin with substitutions of proline residues on its two loops (P37G and P120G) by glycine as well as loop extension in position 120 by three and six glycine residues (P120(3G) and P120(6G)). For all proteins, we measured kinetic denaturation and renaturation curves, calculated rate constants of folding/unfolding, plotted their dependences on the denaturant concentration (chevron plots) and computed free energies of all states of apomyoglobin. The obtained data on the kinetic properties of mutant forms of apomyoglobin allowed us to analyze the effect of flexibility and length of chosen loops on the energy profile of the protein. Specifically, it was demonstrated that the studied mutations destabilized the intermediate state of apomyoglobin as compared to the unfolded state.
apomyoglobin, protein folding, tryptophan fluorescence, stopped-flow experiments, chevron plot, energy profile
1. Gast K., Damaschun H., Misselwitz R., Muller-Frohne M., Zirwer D., Damaschun G. Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate. Eur. Biophys. J., 1994, vol. 23, pp. 297-305.
2. Baryshnikova E.N., Sharapov M.G., Kashparov I.A., Ilyina N.B., Bychkova V.E. Investigation of apomyoglobin stability depending on urea and temperature at two different pH values. J. Mol. Biol., 2005, vol. 39, pp. 292-297.
3. Jennings P.A., Wright P.E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science, 1993, vol. 262, pp. 892-896.
4. Garcia C., Nishimura Ch., Cavagnero S., Dyson H.J., Wright P.E. Changes in the apomyoglobin pathway caused by mutation in the distal histidine residue. Biochemistry, 2000, vol. 39, pp. 11227-11237.
5. Nishimura C., Dyson H.J., Wright P.E. Identification of native and non-native structure in kinetic folding intermediates of apomyoglobin. J. Mol. Biol., 2006, vol. 355, pp. 139-156.
6. Samatova E.N., Katina N.S., Balobanov V.A., Melnik B.S., Dolgikh D.A., Bychkova V.E., Finkelstein A.V. How strong are side chain interactions in the folding intermediate? Protein Sci., 2009, vol. 18, pp. 2152-2159.
7. Samatova E.N., Melnik B.S., Balobanov V.A., Katina N.S., Dolgikh D.A., Semisotnov G.V., Finkelstein A.V., Bychkova V.E. Folding intermediate and folding nucleus for I-N and U-I-N transitions in apomyoglobin: contributions by conserved and nonconserved residues. Biophys J., 2010, vol. 98, pp. 1694-1702.
8. Melnik T.N., Majorina M.A., Larina D.S., Kashparov I.A., Samatova E.N., Glukhov A.S., Melnik B.S. Independent of Their Localization in Protein the Hydrophobic Amino Acid Residues Have No Effect on the Molten Globule State of Apomyoglobin and the Disulfide Bond on the Surface of Apomyoglobin Stabilizes This Intermediate State. Plos.one, 2014, vol. 9, e98645.
9. Mazhorina M.A., Gluhova K.A., Marchenkov V.V., Mel'nik B.S. Vliyanie aminokislotnyh zamen na poverhnosti apomioglobina na energeticheskiy profil' belka. Molekulyarnaya biologiya, 2018, t. 52, № 1, s. 62-72. [Majorina M.A., Glukhova K.A., Marchenkov V.V., Melnik B.S. Effect of substitutions in surface amino acids on energy profile of apomyoglobin. Molecular biology, 2018, vol. 52, no. 1, pp. 62-72 (In Russ.)]
