Chromophore Formation in Green Fluorescent Protein

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• 作者：Brian G. Reid and Gregory C. Flynn
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：June 3, 1997
• 年：1997
• 卷：36
• 期：22
• 页码：6786 - 6791
• 全文大小：207K
• 年卷期：v.36,no.22(June 3, 1997)
• ISSN：1520-4995

文摘

The green fluorescent protein (GFP) from the jellyfishAequorea victoria forms an intrinsicchromophore through cyclization and oxidation of an internal tripeptidemotif [Prasher, D. C., et al. (1992)Gene 111, 229-233; Cody, C. E., et al. (1993)Biochemistry 32, 1212-1218]. We monitored theformationof the chromophore in vitro using the S65T-GFPchromophore mutant. S65T-GFP recovered from inclusionbodies in Escherichia coli lacks the mature chromophore,suggesting that protein destined for inclusionbodies aggregated prior to productive folding. This material wasused to follow the steps leading tochromophore formation. The process of chromophore formation inS65T-GFP was determined to be anordered reaction consisting of three distinct kinetic steps.Protein folding occurs fairly slowly (k_f= 2.44× 10^-3 s^-1) andprior to any chromophore modification. Next, an intermediate stepoccurs that includes,but is not necessarily limited to, cyclization of the tripeptidechromophore motif (k_c = 3.8 ×10^-3s^-1).The final and slow step (k_ox = 1.51 ×10^-4 s^-1) inchromophore formation involves oxidation of thecyclized chromophore. Since the chromophore forms denovo from purified denatured protein and isafirst-order process, we conclude that GFP chromophore formation is anautocatalytic process.