UV resonance Raman determination of protein acid denaturation: selective unfolding of helical segments of horse myoglobin.
We have used UV resonance Raman spectroscopy to study the acid denaturation of horse heart aquometmyoglobin (Mb) between pH 7.5 and 1.5. Raman spectra excited at 206.5 nm are dominated by amide vibrations, which are analyzed by using a new methodology to quantitatively determine the Mb secondary structure. In contrast, the 229-nm Raman spectra are dominated by the Tyr and Trp Raman bands, which are analyzed to examine changes in Tyr and Trp environments, such as exposure to water, hydrogen bonding, and, for Trp, any alterations of the dihedral angle between the Trp ring and its linkage to the protein backbone. We uniquely determined which Mb alpha-helices melt by combining the amide, Tyr, and Trp Raman spectral information with heme absorption spectral information. We calculate that the Mb alpha-helical composition decreases from approximately 80% at neutral pH to approximately 19% below pH 3.5. The Trp Raman cross sections dramatically decrease at low pH to values which indicate that they are fully exposed to water; this result indicates that the A helix melts. The Tyr Raman bands are pH independent, which indicates that the G and H helices around the Tyr residues do not melt. The dramatic heme absorption acid denaturation changes indicate major alterations of the heme pocket and changes in heme binding. These results indicate that the A, B, C, D, E, and F helices melt in a concerted fashion, while the antiparallel G and H helices only partially melt.
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Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
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D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009206:Myocardium / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014466:Ultraviolet Rays
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin