Journal of Molecular Biology
The 2.1 Å Crystal Structure of the Far-red Fluorescent Protein HcRed: Inherent Conformational Flexibility of the Chromophore
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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2019, International Journal of Biological MacromoleculesCitation Excerpt :This also has been observed by the replacement of the equivalent serine with cysteine residue resulted in the fluorescence quantum yield reduction in DsRed, whereas a serine substitution increased fluorescence emission in Rtms5 and cgCP [8,34,35]. The second key residue His-174 in HcRed (Fig. 7c), has also been proposed to introduce the π-stack against the hydroxyphenyl ring particularly in the cis-coplanar conformation for the red-shifted spectra [26]. On the contrary, a leucine residue at this position (Leu-173 in shCP, cjBlue and sgBP) is likely reducing the ring-stacking effect of the stabilization of cis/trans conformations.
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Present address: J. Peterson, Institut fur Physikalische Chemie, Albert-Ludwigs-Universitat Freiburg, Albertstr. 23a, 79104 Freiburg, Germany.