CHARACTERIZATION OF THE FOLDING PATHWAY OF RECOMBINANT HUMAN MACROPHAGE-COLONY STIMULATING-FACTOR-BETA (RHM-CSF-BETA) BY BIS-CYSTEINYL MODIFICATION AND MASS-SPECTROMETRY

Melarsen oxide [p-(4,6-diamino-1,3,5-triazin-2-yl) aminophenylarsonous acid (MEL)], which selectively bridges spatially neighboring bis-cyst einyl residues in (reduced) proteins, was used to trap folding interme diates chemically during 1) time-dependent renaturation of recombinant human macrophage colony-stimulating factor (rhM-CSF); by redox refold ing in vitro; 2) reductive unfolding in the presence of the trapping r eagent; and 3) denaturing unfolding reactions in urea and guanidinium hydrochloride, Characterization of intermediates from folding and unfo lding reactions was performed by electrospray ionization mass spectome try (ESI-MS), In all folding and unfolding reactions a characteristic dimeric intermediate with two attached melarsen oxide (MEL) groups was observed, suggesting that these rhM-CSF beta species were important r efolding intermediates. These intermediates presented a characteristic ''charge structure'' in ESI spectra with a most abundant 26+ charged molecular ion whereas the mature homodimeric rhM-CSF beta showed a mos t abundant 23+ molecular ion, indicating that the final product was mo re compact. The major locations of the two MEL groups were identified by mass spectrometric peptide mapping at cysteine residues C157 and C1 59 from each monomer, Cysteine residues C7 and C90 were minor modifica tion sites. The mass spectrometric results from the in vitro folding r eactions of rhM-CSF beta are in agreement with intrinsic tryptophan fl uorescence measurements and are consistent with the folding pathway th at starts with a fully reduced monomer (R), includes partially folded monomeric intermediates (M) and dimeric intermediates (D), and yields a final product with the native tertiary structure (N): 2R double righ t arrow 2M double right arrow D double right arrow N, Our results show that selective chemical trapping of bis-thiol groups of proteins with MEL permits study of folding pathways by mass spectrometric structure characterization of intermediates with otherwise transient conformati ons. Proteins Suppl, 2:50-62, 1998. (C) 1998 Wiley-Liss, Inc.