Molecular dynamics simulation of the acidic compact state of apomyoglobin from yellowfin tuna

• Published in: Proteins: Structure, Function, and Bioinformatics
• Main Authors: Bismuto, Ettore, Di Maggio, Emiddio, Pleus, Stefan, Sikor, Martin, Röcker, Carlheinz, Nienhaus, G. Ulrich, Lamb, Don C.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2008
• Subjects: Sperm whale
• Online Access: http://dx.doi.org/10.1002/prot.22149; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fprot.22149; https://onlinelibrary.wiley.com/doi/pdf/10.1002/prot.22149

Abstract A molecular model of the acidic compact state of apomyoglobin (A‐state) from yellowfin tuna was obtained using molecular dynamics simulations (MD) by calculating multiple trajectories. To cause partial unfolding within a reasonable amount of CPU time, both an acidic environment (pH 3 and 0.15M NaCl) and a temperature jump to 500 K were needed. Twenty‐five acidic structures of apomyoglobin were generated by MD, 10 of them can be clustered by RMSD in an average structure having a common hydrophobic core as was reported for acidic sperm whale apomyoglobin, with shortened helices A,G,E, and H (the helix A appears to be translated along the sequence). Prolonging the MD runs at 500 K did not cause further substantial unfolding, suggesting that the ensemble of generated structures is indicative of a region of the conformational space accessible to the apoprotein at acidic pH corresponding to a local energy minimum. The comparison of experimentally determined values of specific spectroscopic properties of the apomyoglobin in acidic salt conditions with the expected ones on the basis of the MD generated structures shows a reasonable agreement considering the characteristic uncertainties of both experimental and simulation techniques. We used frequency domain fluorometry, acrylamide fluorescence quenching, and fluorescence correlation spectroscopy together with far UV circular dichroism to estimate the helical content, the Stern–Volmer quenching constant and the radius of gyration of the protein. Tuna apomyoglobin is a single tryptophan protein and thus, interpretation of its intrinsic fluorescence is simpler than for other proteins. The high sensitivity of the applied fluorescence techniques enabled experiments to be performed under very dilute conditions, that is, at concentrations of subnanomolar for the FCS measurements and 6 μM for the other fluorescence measurements. As high concentrations of proteins can strongly affect the association equilibrium among partially unfolded states, fluorescence techniques ...