ISTC 3223 THE STRUCTURE OF THE CHROMOPHORE WITHIN A RED FLUORESCENT PROTEIN FROM ZOANTHUS sp

Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and H and C 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. 17 2. Pletneva N., Pletnev S., Tikhonova T., Popov V., Martynov V., and Pletnev V., (2006) Crystal structure of a red fluorescent protein from Zoanthus, zRFP574, reveals a novel chromophore, Acta Crystallogr D Biol Crystallogr. D62, 527-532. Structure of a red fluorescent protein from Zoanthus, zRFP574, reveals a novel chromophore Nadezhda Pletneva,a Sergei Pletnev,b,c Tamara Tikhonova,d Vladimir Popov,d Vladimir Martynova and Vladimir Pletneva* aShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, Moscow, Russia, bInstitute of Crystallography, Russian Academy of Science, Moscow, Russia, cSynchrotron Radiation Research Section, MCL, National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439, USA, and dBakh Institute of Biochemistry, Russian Academy of Science, Moscow, Russia Correspondence e-mail: [email protected] # 2006 International Union of Crystallography Printed in Denmark – all rights reserved The three-dimensional structure of the red fluorescent protein (RFP) zRFP574 from the button polyp Zoanthus sp. (two dimers per asymmetric unit, 231 _ 4 amino acids) has been determined at 2.4 A ° resolution in space group C2221. The crystal structure, refined to a crystallographic R factor of 0.203 (Rfree = 0.249), adopts the _-barrel fold composed of 11 strands similar to that of the yellow fluorescent protein zYFP538. The zRFP574 chromophore, originating from the protein sequence Asp66-Tyr67-Gly68, has a two-ring structure typical of GFP-like proteins. The bond geometry of residue 66 shows the strong tendency of the corresponding C_ atom to sp2 hybridization as a consequence of N-acylimine bond formation. The zRFP574 chromophore contains the 65–66 cis peptide bond characteristic of red fluorescent proteins. The chromophore phenolic ring adopts a cis conformation coplanar with the imidazolinone ring. The crystallographic study has revealed an unexpected chemical feature of the internal chromophore. A decarboxylated side chain of the chromophore-forming residue Asp66 has been observed in the structure. This additional post-translational modification is likely to play a key role in the bathochromic shift of the zRFP574 spectrum. Received 23 January 2006 Accepted 3 March 2006 PDB Reference: zRFP574, 2fl1, r2fl1sf. 18 List of presentations at conferences and meetings with abstracts 1. Pakhomov A.A., Pletneva N.V., Martynov V.I. The molecular basis for the green to red conversion of the fluorescent protein from Dendronephthya sp. The 30th FEBS Congress and 9th IUBMB Conference “The Protein World”, Budapest, Hungary 2-7 July 2005, The abstract is published in FEBS Journal (2005) 272, Supplement 1, p. 384. The molecular basis for the green to red conversion of the fluorescent protein from Dendronephthya sp. Abstract number: G2-089Pnumber: G2-089P Pakhomov A. A., Pletneva N. V., Martynov V. I. The green fluorescent protein (GFP) from jellyfish Aequorea victoria and its homologs from corals have a broad application in biotechnology as visual reporters for events in living cells. Their applicability in cellular biology is due to autocatalitic synthesis of the chromophore from amino acids inside the protein shell. Thus, these reporters do not require any external agents for the fluorescence appearance. In general the chromophore structure defines spectral properties of the protein whereas alterations in the cromophore environment provides the shift of absorption/emission maxima of no more than some tens of nanometers. In this work we determined the molecular basis of green (emission maximum at 504 nm) to red (emission maximum at 575 nm) conversion of the fluorescent protein from the coral Dendronephthya sp. (DendFP) under UV-irradiation. UV-illumination causes fragmentation of the polypeptide backbone of DendFP, as seen in SDS-PAGE gels. To clarify the chemical nature of this phenomenon DendFP was subjected to proteolytic digestion with trypsin. The chromophorecontaining peptide was isolated from the tryptic digest by HPLC. The structure of the chromopeptide containing the “red” chomophore was determined by ESI, ESI/MS/MS massspectrometry and NMR. The data obtained suggest that the photoinduced green-to-red conversion results in the cleavage of Dend FP polypeptide chain between Leu 64 and His 65 and double bond formation in the side chain of His 65. Consequently, the red shift is explained by the extended p-electron system, which involves the newly formed C=C double bond and imidazole group of His 65. Recently, the analogous photoconversion was found in a fluorescent protein from Trachyphyllia geoffroyi (Kaede). Therefore, DendFP can be attributed to the Kaede subfamily of GFP-like proteins in the classification based on the chromophore structure. Director of the Shemyakin Ovchinnikov Institute of Bioorganic Chemistry, RAS Vadim T. Ivanov Manager of Project # 3223 Vladimir I. Martynov