FTIR studies of recombinant human granulocyte-macrophage colony-stimulating factor in aqueous solutions: secondary structure, disulfide reduction and thermal behavior.
Fourier transform infrared spectroscopy (FTIR) has been used to investigate the secondary structure, disulfide reduction and thermal behavior of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in aqueous solutions. The contributions of amino-acid side-chain groups to the amide I bands of rhGM-CSF in H2O and in D2O solutions were carefully scrutinized, as 40% of the total 127 amino-acid residues of rhGM-CSF is side-chain absorptive (asparagine, glutamine, etc.). The FTIR results indicated that rhGM-CSF is composed of 46% alpha-helix, 7% beta-sheet, 23% turn and 24% loop/irregular structures which are in good agreement with the X-ray diffractional data. Reduction of rhGM-CSF with dithiothreitol caused apparent unfolding of the native conformation followed by the time-dependent increase of beta-aggregation bands which arose at 1622 and 1693 cm(-1) in H2O, 1613 and 1684 cm(-1) in D2O solutions. The result also showed that tertiary structure can change independently of the secondary structure. Thermal denaturation of rhGM-CSF took place at 55 to 70 degrees C and the denatured protein adopted an irregular structure as revealed by the FTIR spectra. The thermal denaturation did not show the formation of intermolecular beta-aggregates which is typical of most thermal denatured proteins. Moreover, it is partly reversible, indicating a special thermal stability of rhGM-CSF.
Jiang
H
H
National Center of Biomedical Analysis, Beijing, People's Republic of China.
Song
Z
Z
Ling
M
M
Yang
S
S
Du
Z
Z
D017666:Deuterium Oxide; D004220:Disulfides; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics; D014867:Water
D004220:Disulfides; D011994:Recombinant Proteins; D012996:Solutions; D014867:Water; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017666:Deuterium Oxide