DISULFIDE-INTACT AND DISULFIDE-REDUCED LYSOZYME IN THE GAS-PHASE - CONFORMATIONS AND PATHWAYS OF FOLDING AND UNFOLDING

The conformations of gaseous lysozyme ions (+5 through +18) produced b y electrospray ionization have been studied in the gas phase using ion mobility mass spectrometry techniques. When solutions containing the disulfide-intact and disulfide-reduced lysozyme are electrosprayed, th e gas-phase ions that are produced have distinctly different collision cross sections. Disulfide-intact ions favor two conformer types: a hi ghly folded conformer with a cross section near that calculated for th e crystal structure and a partially unfolded conformer that is formed when the ions are injected into the drift tube at high injection volta ges. Ions formed from the disulfide-reduced solution have collision cr oss sections that are much larger than any observed for the disulfide- intact protein, showing that these ions are largely unfolded. Gas-phas e proton-transfer reactions in the ion source can be used to favor low er charge states for both solutions. When protons are removed from dis ulfide-intact lysozyme ions, highly folded compact conformations are f avored. Exposing the disulfide-reduced lysozyme ions to proton-transfe r reagents causes the protein to fold up, and several of-the new confo rmations have cross sections that are indistinguishable from those mea sured for the disulfide-intact protein. It appears that an array of ga s-phase folding intermediates or misfolded metastable states are stabl e because of the well-defined interplay between attractive-folding and repulsive-Coulombic interactions.