Structural characterization of the molten globule state of apomyoglobin by limited proteolysis and HPLC-mass spectrometry.
A method to characterize the structural conformation of an acidic molten globule apomyoglobin (apoMb) at pH 4.2 was developed using limited proteolysis and HPLC-mass spectrometry (HPLC-MS). Endoproteinase Glu-C, which has a double maximum activity at pH 4.0 and pH 7.8 toward glutamic acid (Glu), was used as a proteolytic enzyme. Using this method enabled us to compare the proteolytic cleavages of native apoMb (at pH 8.0) and molten globule (at pH 4.2) directly. Only the first cleavage event in each molecule was considered as reflecting original structural information since the original structure of the protein can be altered after the fist cleavage. Structural changes of apoMb in various pH conditions were studied here to elucidate the local helicity of molten globule apoMb. Among 13 Glu sites, only Glu83 and Glu85 in the F-helix were cleaved at pH 8.0, which confirms that only helix F is frayed upon removal of heme group. At acidic molten globule state, rapid cleavages at Glu38, Glu52, Glu54, Glu85, and Glu148 were detected, while the remaining eight sites were protected. Glu6 and Glu18 in the A-helix, and Glu105 in the G-helix were protected due to the helicity of the secondary structures. The cleavage at Glu38 and the protection at Glu41 in the C-helix indicate that the first half of the C-helix is frayed and the second half of the C-helix is structured. Cleavage at both Glu52 and Glu54 in the D-helix proves that the D-helix is disordered. The N-terminal end of the E-helix at Glu59 was protected, and the beginning of the F-helix was protected by aid of the pH-induced C-cap of the E-helix. The cleavage at Glu148 in H suggests that the C-terminal end of the H-helix is disordered. The A-helix and the first half of the B-helix were highly stable.
Kim
Yeoun Jin
YJ
Department of Chemistry, Division of Molecular and Life Sciences, Pohang University of Science and Technology, South Korea.
Kim
Young A
YA
Park
Nokyoung
N
Son
Hyeon S
HS
Kim
Kwang S
KS
Hahn
Jong Hoon
JH
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018698:Glutamic Acid / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008956:Models, Chemical; D009206:Myocardium / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D018698:Glutamic Acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase