Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins

David Buhrke,Neslihan N. Tavraz,Daria M. Shcherbakova,Peter Hildebrandt,Francisco Vélazquez Escobar,Luisa Sauthof,Thomas Friedrich,Marcus Moldenhauer,Vladislav V. Verkhusha

doi:10.1038/s41598-018-38433-2

Abstract

Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.

Highlights

Phytochromes constitute a class of bimodal photoreceptor proteins, which incorporate linear tetrapyrrole cofactors for red light sensing in plants, fungi, cyanobacteria and non-photosynthetic bacteria[1]
In the following) with the Cys residues C15 in the PAS and C249 in the GAF domain (Fig. 2A), we exchanged, first, either of the two cysteines, and second, both cysteines simultaneously for serine, which is non-reactive in chromophore attachment
The resulting topological loop in the protein prevents complete denaturation during SDS treatment, and the protein migrates at a lower apparent mass during electrophoresis. These findings were confirmed by biochemical methods and are in line with corresponding crystallographic data for miRFP670, indicating a mixture of a GAF-only and a double attached species[11]

Summary

Introduction

Phytochromes constitute a class of bimodal photoreceptor proteins, which incorporate linear tetrapyrrole cofactors for red light sensing in plants, fungi, cyanobacteria and non-photosynthetic bacteria[1]. The crystal structure of miRFP67020, an engineered version of the CBD fragment of Rhodopseudomonas palustris phytochrome P1 (RpBphP1), was determined[11] This protein contains two Cys residues in spatial proximity to the vinyl group of the BV ring A (Fig. 2A), one located in the PAS and the other in the GAF domain at homologous positions to the conserved Cys for BphPs, and eukaryotic phytochromes, respectively. Attachment to a secondary Cys residue occurs naturally in diverse cyanobacteriochromes, but in these cases, the secondary Cys is located at different positions in the GAF domain and adds to the A-B or B-C methine bridges[21,22] These findings motivated the present study to determine the binding pattern of BV in iRFP682, another iRFP containing two Cys residues, which was derived from the CBD of RpBphP24.

Methods

Results

Conclusion