Acid-induced unfolding of cytochrome c at different methanol concentrations: electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure.
The acid-induced denaturation of ferricytochrome c (cyt c) was examined in aqueous solutions containing different concentrations of methanol by electrospray ionization mass spectrometry (ESI MS) and optical spectroscopy. Circular dichroism, fluorescence, and absorption spectroscopy show that at a low concentration of methanol (3%) a decrease in pH induces a cooperative unfolding transition at around pH 2.6 that is accompanied by a breakdown of the native secondary and tertiary structure of the protein. In 50% methanol the breakdown of the tertiary structure occurs at around pH 4.0, whereas the alpha-helical content remains largely intact over the whole pH range studied. In ESI MS different protein conformations in solution are monitored by the different charge state distributions they generate during ESI. The ESI mass spectra recorded at near-neutral pH for both methanol concentrations are very similar and show a maximum at (cyt c + 8H+)8+. Despite the different conformations of the protein in solution, the acid-denatured states for the two methanol concentrations also show very similar mass spectra with a maximum at (cyt c + 17H+)17+. This indicates that the charge state distribution generated during ESI is not sensitive to the differences in the secondary structure of the denatured protein. The observed transition from low to high charge states is due to the breakdown of the tertiary structure in both cases. These findings suggest that ESI MS might be a general method to selectively monitor changes in the tertiary structure of proteins.
Konermann
L
L
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Douglas
D J
DJ
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D005470:Fluorometry; D006736:Horses; D006851:Hydrochloric Acid; D013058:Mass Spectrometry; D000432:Methanol; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012996:Solutions; D013053:Spectrophotometry; D014867:Water
D003574:Cytochrome c Group; D012996:Solutions; D014867:Water; D006851:Hydrochloric Acid; D000432:Methanol