The methanol-induced conformational transitions of beta-lactoglobulin, cytochrome c, and ubiquitin at low pH: A study by electrospray ionization massspectrometry

The methanol-induced conformational transitions under acidic conditions for beta -lactoglobulin, cytochrome c, and ubiquitin, representing three diffe rent classes of proteins with beta -sheets, alpha -helices, and both alpha -helices and beta -sheets, respectively, are studied under equilibrium cond itions by electrospray ionization mass spectrometry (ESI-MS). The folding s tates of proteins in solution are monitored by the charge state distributio ns that they produce during ESI and by hydrogen/deuterium (H/D) exchange fo llowed by ESI-MS. The changes in charge state distributions are correlated with earlier studies by optical and other methods which have shown that, in methanol, these proteins form partially unfolded intermediates with induce d ct-helix structure. Intermediate states formed at about 35% methanol conc entration are found to give bimodal charge state distributions. The same ra te of H/D exchange is shown by the two contributions to the bimodal distrib utions. This suggests the intermediates are highly flexible and may consist of a mixture of two or more rapidly interconverting conformers. H/D exchan ge of proteins followed by ESI-MS shows that helical denatured states, popu lated at around 50% methanol concentration, transform into more protected s tructures with further increases in methanol concentration, consistent with previous circular dicroism studies. These more protected structures still produce high charge states in ESI, similar to those of the fully denatured proteins. (C) 2001 American Society for Mass Spectrometry.