Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFbeta).
Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFbeta), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSFbeta) has fast and moderately fast exchangeable sections of amide hydrogens in the alphaA helix, and mostly slow exchanging sections of amide hydrogens in the alphaB, alphaC, and alphaD helices. Most of the amide hydrogens in the loop between the beta1 and beta4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSFalpha than with the average B-factor. The rates of H/D exchange in rhM-CSFbeta appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the alphaD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSFbeta, but not rhM-CSFalpha, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the alphaD helix is due to interaction of the carboxy-terminal tail with this helical segment.
Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, USA.
Xuguang
X
Yan
Heidi
H
Zhang
Jeffrey
J
Watson
Michael I
MI
Schimerlik
Max L
ML
Deinzer
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013997:Time Factors
D004220:Disulfides; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D012997:Solvents; D006859:Hydrogen; D016173:Macrophage Colony-Stimulating Factor; D003903:Deuterium; D003545:Cysteine