Detection of the equilibrium folding intermediate of beta-lactoglobulin in the presence of trifluoroethanol by mass spectrometry.
Nano-electrospray ionization mass spectrometry (nano-ESI-MS) was used to monitor the effect of trifluoroethanol (TFE) on the conformational properties of beta-lactoglobulin (BLG). TFE stabilizes protein secondary structure, particularly alpha-helices. However, it also acts as a denaturant above critical concentrations. In the case of BLG, TFE at low concentrations is known to induce formation of an equilibrium intermediate that contains non-native helical structure. Such an intermediate is thought to form also under physiological conditions, playing a role in BLG folding in vivo by preventing aggregation. This well-characterized system was chosen in order to test species distributions obtained by nano-ESI-MS. BLG spectra at increasing concentrations of TFE at pH 2 indicate transient accumulation of a conformer whose charge-state distribution (CSD) falls between that of the native and that of the denatured protein, indicating that the TFE-induced, partially folded form can be selectively monitored by this technique. The condition of its maximum accumulation corresponds to 16% TFE, in excellent agreement with results from solution experiments. In contrast, titrations with methanol or acetonitrile (ACN) reveal apparent two-state transitions from native to fully unfolded BLG. At 10% TFE, the protein appears to be still fully folded at room temperature but, if unfolding is elicited by the combination with other denaturing agents, e.g. heat or low concentrations of ACN, it proceeds via formation of the intermediate. Thus, TFE can also induce formation of the BLG intermediate in synergism with generic denaturing agents. This study indicates good agreement between ESI-MS and other biophysical methods monitoring protein conformational transitions in the presence of TFE.
Invernizzi
Gaetano
G
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Grandori
Rita
R
D007782:Lactoglobulins / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D017510:Protein Folding; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D007782:Lactoglobulins; D012997:Solvents