title: GFP Loss-of-Function Mutations in Arabidopsis thaliana.
abstract: Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate-induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability.
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Fu
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kanno
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan.
authors.forename: Shih-Chieh
authors.initials: SC
authors.lastname: Liang
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan antoniusmatzke@gate.sinica.edu.tw marjorimatzke@gate.sinica.edu.tw.
authors.forename: Antonius J M
authors.initials: AJ
authors.lastname: Matzke
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan antoniusmatzke@gate.sinica.edu.tw marjorimatzke@gate.sinica.edu.tw.
authors.forename: Marjori
authors.initials: M
authors.lastname: Matzke
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics*; D018389:Codon, Nonsense; D017124:Conserved Sequence; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D030821:Plants, Genetically Modified; D017510:Protein Folding; D055550:Protein Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D018389:Codon, Nonsense; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins
keywords: Arabidopsis thaliana; green fluorescent protein; protein stability; protein structure; reporter gene