Uncoupled analysis of secondary and tertiary protein structure by circular dichroism and electrospray ionization mass spectrometry.
Electrospray ionization mass spectrometry (ESI-MS) applied to protein conformational studies is a powerful new method that seems to provide specific information about protein tertiary structure. In this study, we analyzed the effect of trifluoroethanol (TFE) on a myoglobin peptide and cytochrome c (cyt c) at low pH by circular dichroism (CD) and ESI-MS. These experiments show that coil-to-helix transition per se does not affect ESI mass spectra, confirming that this technique is insensitive to the local conformation of the polypeptidic chain and, rather, reports on the tertiary contacts characterizing different protein conformations. This property makes ESI-MS an excellent method, complementary to CD, for the characterization of protein conformational changes. Fluorinated alcohols have been suggested to induce molten globule formation in acid-unfolded cyt c. The experiments described here show that TFE does not induce major changes in the ESI mass spectrum of cyt c at pH 2.2, indicating that no stabilization of compact, globular structures is detectable under the conditions employed. On the other hand, even low concentrations of TFE (2-5%) are shown to destabilize the folded state of the protein around the mid-point of its acid-induced unfolding transition.
Grandori
Rita
R
Institute of Chemistry, Johannes Kepler University, Altenbergerstrasse 69, A-4040 Linz, Austria. rita.grandori@jk.uni-linz.ac.at
Matecko
Irena
I
Müller
Norbert
N
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000032:analysis* / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry; D036103:Nanotechnology / Q000379:methods; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014270:Trifluoroethanol / Q000737:chemistry
D003574:Cytochrome c Group; D009211:Myoglobin; D014270:Trifluoroethanol