Probing the structure of human growth hormone by limited proteolysis.
Digestion of human growth hormone (hGH) with the Glu-specific protease from Staphylococcus aureus V8 was performed at 20-22 degrees C or 37 degrees C at a 1:20 ratio (by weight) at pH 7.8 with or without 0.2% SDS. There are 14 Glu-residues evenly distributed along the polypeptide chain of hGH as possible sites of proteolytic cleavage of V8-protease. The pattern of fragmentation of hGH was analyzed by electrophoresis and reversed-phase HPLC, and the identity of the proteolytic fragments isolated to homogeneity was established by their partial sequencing and amino acid analysis after acid hydrolysis. Kinetic analysis of the proteolytic digestion process allowed to establish that initial nicking of the protein occurs at Glu33 and subsequently at Glu56 and Glu66. Much slower cleavages occur at Glu30 and Glu186. These cleavage sites are located at chain loops in the hGH molecule, and in particular outside the helical segments of the four-helix bundle of the crystal structure of hGH. Fragments 1-33 and 67-191 comprising entirely the N-terminal helix and the three C-terminal helices of hGH, respectively, were isolated to homogeneity in amounts useful for subsequent conformational and functional studies. The results of this study and of previous ones [Li, C.H. (1982) Mol. Cell. Biochem. 46, 31-41] describing limited proteolysis of hGH by various proteases have been interpreted on the basis of the three-dimensional structure and dynamics of hGH. Overall, it is shown that proteolytic enzymes preferentially cleave hGH at exposed and flexible loops only, thus emphasizing the fact that proteases can be used as reliable probes of protein structure and dynamics.
Polverino de Laureto
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CRIBI Biotechnology Centre, University of Padua, Italy.
Toma
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Tonon
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Fontana
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A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000032:analysis; D013006:Growth Hormone / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism; D013696:Temperature
D000596:Amino Acids; D010446:Peptide Fragments; D018698:Glutamic Acid; D013006:Growth Hormone; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase