Structural Basis of Enhanced Photoconversion Yield in Green Fluorescent Protein-like Protein Dendra2

Abstract

Dendra2 is an engineered, monomeric GFP-like protein that belongs to a subclass of fluorescent proteins undergoing irreversible photoconversion from a green- to a red-emitting state upon exposure to purple-blue light. This photoinduced process occurs only in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized pi-electron system. We have measured the X-ray structure of the green species of Dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. The structure, which is very similar to those reported for the closely related proteins EosFP and Kaede, revealed a local structural change involving mainly Arg66 and a water molecule W4, which are part of a charged and hydrogen-bonded cluster of amino acids and water molecules next to the chromophore. Unlike in EosFP and Kaede, Arg66 of Dendra2 does not contribute to negative charge stabilization on the imidazolinone ring by hydrogen bonding to the imidazolinone carbonyl. This structural change may explain the blue shift of the absorption and emission bands, as well as the markedly higher pKs of the hydroxyphenyl moiety of the chromophore, which were determined as 7.1 and 7.5 for the green and red species, respectively. The action spectrum of photoconversion coincides with the absorption band of the neutral species. Consequently, its 20-fold enhancement in Dendra2 at physiological pH accounts for the higher photoconversion yield of this protein as compared to EosFP.

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states

“…X-ray structures have been reported for a number of irreversible PAFPs, including those that change their fluorescence from green to red upon irradiation with violet light such as EosFP (3), its derivative IrisFP (4), Kaede (5), KikGR (6), and Dendra2 (7). These PAFPs share the same His-Tyr-Gly chromophore-forming tripeptide and the same mechanism of photoactivation.…”

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

“…Photoconversion/photoswitching proceeds usually via the neutral chromophore that absorbs at shorter wavelengths as the anionic form (32,66). Consequently, shifting of the ground state equilibrium towards the neutral form can increase the conversion/switching efficiency and allows to reduce the dose of irradiation required to achieve the desired effect (104,105). However, the increase in conversion efficiency will come at the expense of brightness that is lowered along with the concentration of the anionic chromophore.…”

The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

“…All of the programs used for structure determination and refinement are included in the CCP4 suite (Collaborative Computational Project, Number 4, 1994). Molecular replacement was carried out with the program MOLREP (Vagin & Teplyakov, 2000) using the coordinates of Dendra2 (76% sequence identity; PDB code 2vzx; Adam et al, 2009) as the search model. The structure was built and refined using ARP/wARP (Lamzin & Wilson, 1993), REFMAC5 (Murshudov et al, 1997) and Coot (Emsley & Cowtan, 2004).…”

“…mAG is highly homologous to Dendra2 (Adam et al, 2009), Dronpa (on-state, Andresen et al, 2007;off-state, Stiel et al, 2007), KikG (Tsutsui et al, 2005) and Kaede (Hayashi et al, 2007). Their fluorescent properties, sequence identities, PDB codes, chromophores and r.m.s.d.…”

“…To reveal this basis, we have determined the 2.2 Å crystal structure of mAG and compared it with the crystal structures of highly homologous proteins with various fluorescent properties: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein with an HYG chromophore; Adam et al, 2009), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein with a QYG chromophore; Andresen et al, 2007;Stiel et al, 2007), KikG (a tetrameric green-emitting fluorescent protein with a DYG chromophore; Tsutsui et al, 2005) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein with an HYG chromophore; Hayashi et al, 2007) as well as avGFP (Ormö et al, 1996). Our structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66, and Thr69 are likely to be required for the stable green emission of mAG.…”