Structure and reactivity of the chromophore of a GFP-like chromoprotein from Condylactis gigantea.
Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and 1H and 13C NMR of the chromopeptide reveal that the protein contains a chromophore with a chemical structure identical to that of the red fluorescent protein from Discosoma sp. A single A63G substitution demonstrates that the nature of the first amino acid of the XYG chromophore-forming sequence is dispensable for the chromoprotein red shift development. It has been recently proposed that post-translational reactions at the acylimine, a chemical group that accounts for the red fluorescence, might be an additional source of spectral diversity of proteins homologous to the Aequorea victoria green fluorescent protein (GFP). We have examined the reactivity of the chromophore acylimine group within the C. gigantea purple chromoprotein. Like other proteins with the acylimine-modified chromophore, the purple chromoprotein suffers a hypsochromic spectral shift to the GFP-like absorbance (386 nm) upon mild denaturation. NMR analysis of the chromopeptide suggests this hypsochromic spectral shift is due to H2O addition across the C=N bond of the acylimine. However, unlike the red fluorescent protein from Discosoma sp., denatured under harsh conditions, the wild-type chromoprotein exhibits only slight fragmentation, which is induced by complete hydrolysis of the acylimine. A model suggesting the influence of the amino acid X side chain on protein fragmentation is presented.
Pakhomov
Alexey A
AA
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Pletneva
Nadezhda V
NV
Balashova
Tamara A
TA
Martynov
Vladimir I
VI
D000818:Animals; D037421:Anthozoa / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D049452:Green Fluorescent Proteins