Femtosecond Studies of the Initial Events in the Photocycle of Photoactive Yellow Protein (PYP)

Horn, Matthew A.; Gruetzmacher, Julie A.; Kim, Jeongho; Choi, Seung‐Eng; Anderson, Spencer; Moffat, Keith; Scherer, Norbert F.

doi:10.1002/3527600183.ch23

Cited by 3 publications

(2 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast to fluorescent proteins, PYP and rhodopsin have exceedingly low fluorescence quantum yields [2 × 10 −3 (64) and 1 × 10 −5 (51), respectively], while exhibiting excellent cis-trans isomerization efficiencies [quantum yields 35% (65) and 65% (66), respectively]. Early studies suggest their similarities (52,67,68): they have steep Franck-Condon regions (i.e., strong vibronic coupling along the reaction coordinate) and either small or no excited-state barriers, allowing the excited population to rush down to the conical intersection without much impediment, and their conical intersections are relatively more peaked than sloped to increase isomerization quantum yields while suppressing competing internal conversion (45,46,53,69). Although fluorescent proteins and photosensory proteins have drastically different fluorescence and isomerization quantum efficiencies from one another, they can all be described with the same type of PES topology, suggesting that we can learn from photosensory proteins to improve the photodissociation of split GFPs.…”

Section: Resultsmentioning

confidence: 99%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.split GFP | potential energy surface | photodissociation | cis-trans isomerization | photosensory protein O ptical techniques for investigating biological processes in vivo can achieve subcellular spatial and millisecond temporal resolution by using genetically encoded light-responsive proteins (1). Photoactivatable proteins are used as either imaging tools, such as reversibly photoswitchable fluorescent proteins (RSFPs), with fluorescence that can be modulated by light (2) or optogenetic switches that convert light input into physiological outputs, such as channelrhodopsins, which can regulate ion flow through membranes in response to light (3). Split GFPs have been widely applied for imaging as fluorescent reporters of cellular processes, because they are small (∼25 kDa), are stable in cytosol, produce chromophores autocatalytically, and are amenable to mutation and circular permutation (4). Typically, the protein is expressed as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another, offering readouts of protein-protein colocalizaton with low background and high specificity (5). However, this technique can generate misleading results, because the GFP complexes, after being formed and fluorescing, are bound irreversibly, which can be highly perturbative to the processes being studied, especially if the protein interaction being probed is ordinarily reversible (6). Although split GFP complexes essentially do not dissocia...

show abstract

Section: Resultsmentioning

confidence: 99%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…We selected wt PYP and the E46H, E46K, and E46Y mutants for this analysis because they span the entire range of Φ fl values. The pumpprobe measurements were performed with detection in the region 400-515 nm, since the electronically excited state has an absorbance band in this region (33,34). The resulting time traces (Fig.…”

Section: Correlating Fluorescence Quantum Yield and Excited State Lifmentioning

confidence: 99%

Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface

Philip

Nome

Papadantonakis

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Protein-chromophore interactions in photoreceptors often shift the chromophore absorbance maximum to a biologically relevant spectral region. A fundamental question regarding such spectral tuning effects is how the electronic ground state S 0 and excited state S 1 are modified by the protein. It is widely assumed that changes in energy gap between S 0 and S 1 are the main factor in biological spectral tuning. We report a generally applicable approach to determine if a specific residue modulates the energy gap, or if it alters the equilibrium nuclear geometry or width of the energy surfaces. This approach uses the effects that changes in these three parameters have on the absorbance and fluorescence emission spectra of mutants. We apply this strategy to a set of mutants of photoactive yellow protein (PYP) containing all 20 side chains at active site residue 46. While the mutants exhibit significant variation in both the position and width of their absorbance spectra, the fluorescence emission spectra are largely unchanged. This provides strong evidence against a major role for changes in energy gap in the spectral tuning of these mutants and reveals a change in the width of the S 1 energy surface. We determined the excited state lifetime of selected mutants and the observed correlation between the fluorescence quantum yield and lifetime shows that the fluorescence spectra are representative of the energy surfaces of the mutants. These results reveal that residue 46 tunes the absorbance spectrum of PYP largely by modulating the width of the S 1 energy surface.photoreceptor | protein-chromophore interactions | wavelength regulation L ight-driven proteins, consisting of a protein-chromophore complex, employ two general strategies to optimize the biological effectiveness of the position of their absorbance spectra. First, covalent modifications of a chromophore can shift its absorbance maximum λ abs max . An important example is the strong red-shift in the absorbance spectrum of bacteriochlorophyll compared to plant chlorophyll (1). Second, specific protein-chromophore interactions can shift the absorbance spectrum of the protein-bound chromophore. A classic example of this spectral tuning phenomenon is color vision in vertebrates (2, 3): the same retinal chromophore can absorb in the blue, green, and red, depending on the amino acid sequence of the rhodopsin to which it is bound. Spectral tuning provides an example of protein-ligand interactions that tune the properties of the cofactor to biologically relevant values.Two main approaches have been used to unravel the factors involved in spectral tuning: determining (i) which amino acids in the protein contribute to spectral tuning, and (ii) what type of protein-chromophore interactions cause spectral tuning. Interactions that alter the degree of charge delocalization over the chromophore are of particular importance. These two issues have been explored extensively in animal visual rhodopsins and the archaeal rhodopsins (4-7). Here we study spectral tuning in a more r...

show abstract

Polarized transient absorption studies of the initial isomerization dynamics of photoactive yellow protein, its E46Q derivative, and model chromophore compounds

Larsen

Stokkum

Vengris

et al. 2002

The Thirteenth International Conference on Ultrafast Phenomena

View full text Add to dashboard Cite

show abstract

Femtosecond Studies of the Initial Events in the Photocycle of Photoactive Yellow Protein (PYP)

Cited by 3 publications

References 4 publications

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface

Polarized transient absorption studies of the initial isomerization dynamics of photoactive yellow protein, its E46Q derivative, and model chromophore compounds

Contact Info

Product

Resources

About