Understanding GFP Chromophore Biosynthesis:  Controlling Backbone Cyclization and Modifying Post-translational Chemistry<sup>,</sup>

Barondeau, D.P.; Kassmann, C.J.; Tainer, John A.; Getzoff, Elizabeth D.

doi:10.1021/bi0479205

Cited by 79 publications

(123 citation statements)

References 37 publications

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“…A similar reversible hydration reaction was postulated, although controversially discussed, to occur during the chromophore formation of GFP [27][28][29] . This might suggest that the light-induced reversible switching of Dreiklang is based on a molecular reaction that is possibly occurring during chromophore maturation of some fluorescent proteins.…”

Section: To Further Confirm This Light-induced Chemical Modificationmentioning

confidence: 91%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

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VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

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Section: To Further Confirm This Light-induced Chemical Modificationmentioning

confidence: 91%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

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“…6). [46][47][48][49][50][51] A number of crystal structures are known for the conformation of the precyclized state, and these structures are of two distinct types. In one type of precyclized structure, an n!p* interaction exists between the main-chain carbonyl group of residue 62 and the chromophore [d ¼ 3.0 Å , y ¼ 97 ; Fig.…”

Section: -27mentioning

confidence: 99%

A conserved interaction with the chromophore of fluorescent proteins

2011

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The chromophore of fluorescent proteins, including the green fluorescent protein (GFP), contains a highly conjugated imidazolidinone ring. In many fluorescent proteins, the carbonyl group of the imidazolidinone ring engages in a hydrogen bond with the side chain of an arginine residue. Prior studies have indicated that such an electrophilic carbonyl group in a protein often accepts electron density from a main-chain oxygen. A survey of high-resolution structures of fluorescent proteins indicates that electron lone pairs of a main-chain oxygen-Thr62 in GFP-donate electron density into an antibonding orbital of the imidazolidinone carbonyl group. This nfip* electron delocalization prevents structural distortion during chromophore excitation that could otherwise lead to fluorescence quenching. In addition, this interaction is present in on-pathway intermediates leading to the chromophore, and thus could direct its biogenesis. Accordingly, this nfip* interaction merits inclusion in computational and photophysical analyses of the chromophore, and in speculations about the molecular evolution of fluorescent proteins.

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“…13 Indeed, the crystallographic analysis of the so-called Ser65Ala and Tyr66Ser GFPhal variants revealed a planar five-membered MIO. It was then hypothesized that the GFP and HAL scaffolds promote backbone cyclization by aligning in close proximity the Gly(i) amide lone pair and the π orbital of the residue i−2 carbonyl and preventing the involvement of these main chain groups in intramolecular hydrogen bonding interactions in the precursor state.…”

Section: [Scheme 1]mentioning

confidence: 99%

“…13 These authors found, by means of UV-vis spectroscopic measurements, that engineered GFPhal variants consist of a mixed population containing aromatic and nonaromatic MIO species, which are interchangeable throughout a quasi-reversible dehydration/hydration reaction.…”

Section: [Scheme 2]mentioning

confidence: 99%

“…10 Once the presence and structure of MIO were demonstrated, several laboratories tried to gain insight into both the biogenesis of this novel prosthetic group and the details of the catalytic mechanism of these enzymes. Knowledge about the former unique process was obtained over the years by (i) biochemical, biophysical and X-ray crystallographic analyses of several wild-type 7 and site-point variants of HAL 11 and PAL enzymes 12 , (ii) 'grafting' of the MIO-forming tripeptide into the GFP sequence by means of structure-guided protein engineering, 13 and (iii) quantum mechanical (QM) calculations using reduced model systems. 14 In their seminal report on the crystal structure of mature Pp-HAL, Schulz et al 7 postulated a mechanism (Scheme 1, A) that was analogous to that proposed for fluorophore biosynthesis in Aequorea victoria GFP (Av-GFP) by Tsien and co-workers a few years earlier.…”

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confidence: 99%

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Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

et al. 2016

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A 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) electrophilic moiety is post-translationally and autocatalytically generated in homotetrameric histidine ammonia-lyase (HAL) and other enzymes containing the tripeptide Ala-Ser-Gly in a suitably positioned loop. The backbone cyclization step is identical to that taking place during fluorophore formation in green fluorescent protein from the tripeptide Ser-Tyr-Gly, but dehydration, rather than dehydrogenation by molecular oxygen, is the reaction that gives rise to the mature MIO ring system. To gain additional insight into this unique process and shed light on some still unresolved issues, we have made use of extensive molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations implementing the self-consistent charge density functional tight-binding method on a fully solvated tetramer of Pseudomonas putida HAL. Our results strongly support the idea that mechanical compression of the reacting loop by neighboring protein residues in the precursor state is absolutely required to prevent formation of inhibitory main-chain hydrogen bonds and to enforce proper alignment of donor and acceptor orbitals for bond creation. The consideration of the protein environment in our computations shows that water molecules, which have been mostly neglected in previous theoretical work, play a highly relevant role in the reaction mechanism and, more importantly, that backbone cyclization resulting from the nucleophilic attack of the Gly amide lone pair on the π* orbital of the Ala carbonyl precedes side-chain dehydration of the central serine.

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Understanding GFP Chromophore Biosynthesis: Controlling Backbone Cyclization and Modifying Post-translational Chemistry^,

Cited by 79 publications

References 37 publications

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

A conserved interaction with the chromophore of fluorescent proteins

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

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Understanding GFP Chromophore Biosynthesis: Controlling Backbone Cyclization and Modifying Post-translational Chemistry,

Cited by 79 publications

References 37 publications

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

A conserved interaction with the chromophore of fluorescent proteins

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

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Understanding GFP Chromophore Biosynthesis: Controlling Backbone Cyclization and Modifying Post-translational Chemistry^,