Characterization of the folding pathway of recombinant human macrophage‐colony stimulating‐factor β (rhM‐CSF β) by bis‐cysteinyl modification and mass spectrometry

Abstract

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.

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

“…In gel enzymatic digestion 28 results in mixtures of peptides that carry structural information from the parent protein. Mass spectrometry in combination with proteolysis is well suited for the study of structural details of proteins 29–31. Here we show that at least the short splice variant of sCD21, without exon 11, circulates in plasma and that the N‐terminal amino acid of the molecule is non‐modified I21, as anticipated for the processed CD21 lacking the leader peptide.…”

Protein‐folding kinetics and mechanisms studied by pulse‐labeling and mass spectrometry

“…Happersberger et al (1998) studied the oxidative refolding of recombinant human macrophage‐colony stimulating‐factor β, which occurs on the time‐scale of tens of hours. The reduced form of this protein adopts an unfolded monomeric structure, whereas the native state is a dimer that is stabilized by intra‐ and intermolecular disulfide bonds.…”

“…Most likely, this charge stabilization occurs through intramolecular solvation and through the greater spatial separation of individual charges, both of which serve to reduce electrostatic repulsion. In this sense, the ESI charge‐state distribution may be considered to be a probe of the overall “compactness” of a protein in solution (Happersberger et al, 1998; Miranker, 2000; Gumerov et al, 2002; Kaltashov & Eyles, 2002; Kim et al, 2002). Konermann & Douglas (1997) observed that changes of the cyt c charge‐state distribution selectively reflect alterations of the protein's tertiary structure, but remain insensitive to secondary structure changes.…”

Mass Spectrometry Unravels Disulfide Bond Formation as the Mechanism That Activates a Molecular Chaperone

“…On target reduction in the presence of 4-hydroxy-␣-cyano-cinnamic acid (HCCA) matrix was carried out with the Lys-C-derived Hsp33 peptides after MALDI-MS peptide mapping (see below) according to published procedures (8,10,11).…”

“…Nano-ESI-MS was performed with a Vestec-201A quadrupole mass spectrometer as described previously (10). Mass calibration was performed with the 8ϩ to 12ϩ charged ions of hen egg white lysozyme (M r 14305.1), and raw data were analyzed using a Vector-2 data system (Teknivent).…”