The 2.1 Å Crystal Structure of the Far-red Fluorescent Protein HcRed: Inherent Conformational Flexibility of the Chromophore

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We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1 Å resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu64-Tyr65-Gly66, was observed to adopt both a cis coplanar and a trans non-coplanar conformation. As a result of these two conformations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the β-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible.

Introduction

All-protein chromophores are found in a variety of marine organisms and possess spectral properties covering the entire visual range of wavelengths including highly fluorescent proteins (FPs), and the intensely coloured but non-fluorescent chromoproteins (CPs).1, 2, 3, 4

The chromophore responsible for light absorption and its fluorescence properties in FPs and CPs arises from an extended conjugated π-system that comprises a cyclic tri-peptide structure. This chromophore forms spontaneously inside a characteristic 11-stranded β-barrel from the covalent rearrangement of three consecutive amino acid residues (XXG). The maturation of the chromophore is autocatalytic and is solely dependent on the presence of molecular oxygen.

Detailed investigations of the green fluorescent protein (GFP)-like family have revealed some novel aspects of protein chemistry. Aside from the unique cyclisation events leading to formation of the chromophore,5 it has been recognised that the spectral properties of the chromophore in certain proteins can be altered by novel post-translational modifications. For example, photoconversion of GFP involves decarboxylation of a glutamate residue close to the chromophore,6 whilst photoconversion of the “Kaede” protein involves an unconventional cleavage of a peptide bond adjacent to the chromophore.7 Maturation of the yellow fluorescent protein zFP538 from Zoanthus sp. involves the nucleophilic attack of a transient acylimine intermediate, resulting in the formation of a three-ring chromophore with an associated polypeptide cleavage between Phe-65 C′ and Lys-66Nα.8

In addition, chromophores within the GFP-like family can adopt long-lived alternative conformations despite sharing similar chromophore sequences. For example, the chromophore (Met63-Tyr64-Gly65) in the highly far-red fluorescent protein (QY, 0.45), eqFP611, adopts a trans coplanar conformation,9 the chromophore (Gln66-Tyr67-Gly68) in the non-fluorescent CP (QY<0.0002), Rtms5, adopts a trans non-coplanar conformation,10 whilst an identical chromophore sequence in the highly fluorescent DsRed (QY, 0.70) adopts a cis coplanar conformation.11 Recently a trans non-coplanar chromophore (Met63-Tyr64-Gly65) was observed for a protein derived from the chromoprotein asCP, the kindling protein KFP1.12 Cis–trans isomerisation of the chromphore has been suggested to be a key feature of the mechanism of kindling of certain chromproteins.13 These observations suggest, in part, the non-coplanar conformation contributes to the low quantum yield of chromoproteins and the coplanar conformation contributes to fluorescence of fluorescent proteins.

Non-fluorescent chromoproteins represent a source of far-red (>620 nm) fluorescent proteins; however, they generally exhibit very low quantum yield.10, 14, 15 Specific amino acid substitutions around the chromophore are able to convert such non-fluorescent chromoproteins into higher quantum yield fluorescent proteins with far-red shifted emission spectra. In order to further explore the inter-relationship between chromophore conformation and fluorescence in this GFP-like family, we determined the crystal structure of HcRed, a chromoprotein isolated from Heteractis crispa that was converted to a far-red fluorescent protein (Emmax=645 nm; QY, 0.05), to 2.1 Å resolution. The chromophore was observed to adopt both a cis coplanar and a trans non-coplanar conformation. These observations together with a quantum chemical modelling of the alternative chromophore conformations are consistent with the cis conformation being related to increased fluorescence of engineered HcRed protein. Further stabilization of the cis conformation should result in enhanced fluorescent properties of HcRed.

Section snippets

Spectral properties of HcRed

Chromoproteins and fluorescent proteins undergo a number of temperature-sensitive post-translational covalent rearrangements (termed maturation) that lead to the formation of the chromophore. Maturation of HcRed appears to follow a pattern similar to the chromoproteins cgigCP16 and Rtms5 (M.P., unpublished results). The progress of maturation can be monitored by following the absorbance spectrum of these proteins. To minimise maturation of the chromophore, freshly expressed HcRed was rapidly

Discussion

The crystal structure of the engineered, far-red fluorescent HcRed has been determined to 2.1 Å resolution. A dimeric oligomeric state of HcRed was observed in solution, as well as in the crystal lattice. One of the hcCP substitutions that promoted dimer formation resides within the oligomeric interface of the tetrameric GFP-like proteins.

The HcRed structure has revealed that the tripeptide chromophore, Glu64-Tyr65-Gly66, is surprisingly mobile within the interior of the β-can (Figure 4). The

Cloning

A DNA cassette encoding HcRed was retrieved by PCR from pHcRed1-N1 (BD Biosciences) using the oligonucleotide primer pair 5′taggatccatcgccaccatggtgagcggcctg/5′ atagtttagcggccgctcagtgatcagagttggccttctcgggcag and cloned into the BamHI/NotI site of pQE10N. An expression cassette encoding enhanced green fluorescent protein (yEGFP3)18 was excised from the yeast expression vector pAS1NB∷YEGFP3L32 and cloned into pQE10N. pQE10N is a derivative of pQE10 (Qiagen) modified to contain a NotI in the

Acknowledgements

This work was supported by a Monash University small grant. P.W. is supported by a Monash University PhD scholarship. J.R. is supported by a Wellcome Trust Senior Research Fellowship in Biomedical Science in Australia.

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    Present address: J. Peterson, Institut fur Physikalische Chemie, Albert-Ludwigs-Universitat Freiburg, Albertstr. 23a, 79104 Freiburg, Germany.

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