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-cysteinyl residues in (reduced) proteins, was used to trap folding intermediates chemically during 1) time-dependent renaturation of recombinant human macrophage colony-stimulating factor (rhM-CSF); by redox refolding in vitro; 2) reductive unfolding in the presence of the trapping reagent; and 3) denaturing unfolding reactions in urea and guanidinium hydrochloride. Characterization of intermediates from folding and unfolding reactions was performed by electrospray ionization mass spectometry (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 refolding 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 most abundant 23+ molecular ion, indicating that the final product was more compact. The major locations of the two MEL groups were identified by mass spectrometric peptide mapping at cysteine residues C157 and C159 from each monomer. Cysteine residues C7 and C90 were minor modification sites. The mass spectrometric results from the in vitro folding reactions of rhM-CSF beta are in agreement with intrinsic tryptophan fluorescence measurements and are consistent with the folding pathway that 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 ==> 2M ==> D ==> 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 conformations.