Cynthia Cowgill

Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) 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 β 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 β 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 β 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. Proteins Suppl. 2:50–62, 1998.

Structural characterization of monomeric folding intermediates of recombinant human macrophage-colony stimulating factor β (rhM-CSFβ) by chemical trapping, chromatographic separation and mass spectrometric peptide mapping

We have developed a strategy for the characterization of protein folding intermediates that combines selective modification of bis-cysteinyl thiol groups with melarsen oxide (MEL), chromatographic separation and mass spectrometric characterization of the resulting protein derivatives. In the unfolding reaction of recombinant human macrophage-colony stimulating-factor beta (rhM-CSFbeta) we observed monomeric M.4MEL and dimeric D.2MEL intermediates. The major locations of the MEL groups in D.2MEL were at C157 and C159. In M.4MEL, MEL groups were predominantly located at C31 and C102. These results indicate the presence of highly structured dimeric and monomeric intermediates. In the completely reduced R.4MEL derivative, MEL groups were distributed such that the smallest ring structures resulted.