tailieunhanh - Báo cáo khóa học: Vertical-scanning mutagenesis of amino acids in a model N-myristoylation motif reveals the major amino-terminal sequence requirements for protein N-myristoylation

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of thesemutants to protein N-myristoylation was evaluated by metabolic labe-ling in anin vitrotranslation systemusing rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAA AAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation | Eur. J. Biochem. 271 863-874 2004 FEBS 2004 doi Vertical-scanning mutagenesis of amino acids in a model N-myristoylation motif reveals the major amino-terminal sequence requirements for protein N-myristoylation Toshihiko Utsumi Kengo Nakano Takeshi Funakoshi Yoshiyuki Kayano Sayaka Nakao Nagisa Sakurai Hiroyuki Iwata1 and Rumi Ishisaka Department of Biological Chemistry and -Department of Veterinary Medicine Faculty of Agriculture Yamaguchi University Yamaguchi Japan In order to determine the amino-terminal sequence requirements for protein N-myristoylation site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor TNF mutants as model substrate proteins. Subsequently the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAA AAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the ma or sequence requirements for protein N-myristoyla-tion the combination of amino acids at position 3 and 6 constitutes a ma or determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6 -- amino acids Gly Ala Ser Cys Thi . Vai An Leu lie Gln His were permitted at position 3 to direct efficient protein N-myristoylation. In this case the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6 only 3 amino acids Ala Asn Gln were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which .

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