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1 Department of Chemistry, Oregon State University, Corvallis, Oregon 97330, USA
2 Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97330, USA
3 Present address: Department of Biomolecular Mass Spectrometry Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CA, Utrecht, The Netherlands.
Reprint requests to: Dr. Max Deinzer, Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA; e-mail: max.deinzer{at}orst.edu; fax: (541) 737-0497.
Hydrogen deuterium exchange, monitored by electrospray ionization mass spectrometry, has been employed to characterize structural features of a derivative of recombinant human macrophage colony stimulating factor beta (rhm-CSFß) in which two of the nine disulfide bridges (Cys157/Cys159–Cys`157/Cys`159) were selectively reduced and alkylated. Removal of these two disulfide bridges did not affect the biological activity of the protein. Similarities between CD and fluorescence spectra for rhm-CSFß and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three-dimensional structure of rhm-CSFß. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm-CSFß. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm-CSFß.
Keywords: Macrophage colony stimulating factor ß; disulfide bond; hydrogen deuterium exchange; electrospray ionization mass spectrometry
Abbreviations: aa, amino acid • CD, circular dichroism • CDAP, 1-cyano-4-dimethylaminopyridinium fluoroborate • CID, collision-induced dissociation • CN, cyanylation • rhm-CSFß, recombinant human macrophage stimulating factor ß • cDNA, complementary DNA • E. coli, Escherichia coli • H/D-ESI-MS, hydrogen deuterium exchange electrospray ionization mass spectrometry • kD, kilodaltons • MS/MS, tandem mass spectrometry • NMR, nuclear magnetic resonance • RP-HPLC, reversed-phase high-performance liquid chromatography • TCEP, Tris(2-carboxyethyl)phosphine
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