PROTEIN CONFORMATIONAL-CHANGES DETERMINED BY MATRIX-ASSISTED LASER-DESORPTION MASS-SPECTROMETRY

It is shown that simultaneously to the unfolding of hen egg white lyso zyme and horse heart cytochrome c the sequential conformational change s and molten globule states can be detected by the combination of prot eolysis and matrix-assisted laser desorption/ionization mass spectrome try (MALDI-MS), This is demonstrated by the differences among the prod ucts and the time courses of native lysozyme as well as those unfolded in 1 and. 3 M guanidine hydrochloride (GuHCl) when they were proteoly zed by proteinase K and analyzed by MALDI-MS. Due to the absence of di sulfide bonds in the cytochrome c molecule, it is more sensitive to th e disturbance of the denaturant. The partially unfolded state as detec ted at low concentrations of guanidine hydrochloride in our experiment resemble the molten globule state, One of the unique properties of th e method described herein is to measure directly the peptide fragment liberated hom proteolysis of the protein, It, allows the identificatio n of the sensitive sites susceptible to denaturation, which are subseq uently cleaved by proteinase K proteolysis. (C) 1998 Academic Press.