scholarly journals Structural and Spectroscopic Characterization of Photoactive Yellow Protein and Photoswitchable Fluorescent Protein Constructs Containing Heavy Atoms

2020 ◽  
Author(s):  
Matthew Romei ◽  
Chi-Yun Lin ◽  
Steven Boxer
Keyword(s):  
Electron Diffraction ◽  
Fluorescent Protein ◽  
Heavy Atom ◽  
Spectroscopic Studies ◽  
Photoactive Yellow Protein ◽  
Structural Rearrangements ◽  
Time Resolved ◽  
Heavy Atoms ◽  
Protein Constructs

Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore’s photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-the-art time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization.

scholarly journals Structural and Spectroscopic Characterization of Photoactive Yellow Protein and Photoswitchable Fluorescent Protein Constructs Containing Heavy Atoms

2020 ◽  
Author(s):  
Matthew Romei ◽  
Chi-Yun Lin ◽  
Steven Boxer
Keyword(s):  
Electron Diffraction ◽  
Fluorescent Protein ◽  
Heavy Atom ◽  
Spectroscopic Studies ◽  
Photoactive Yellow Protein ◽  
Structural Rearrangements ◽  
Time Resolved ◽  
Heavy Atoms ◽  
Protein Constructs

Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore’s photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-the-art time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization.

Electron Diffraction of Platinum Labelled Bacteriorhodopsin

1980 ◽  
Vol 38 ◽  
pp. 34-35
Author(s):  
M. E. Dumont ◽  
J. W. Wiggins ◽  
S. B. Hayward
Keyword(s):  
Amino Acids ◽  
High Resolution ◽  
Electron Diffraction ◽  
Structural Information ◽  
Heavy Atom ◽  
Halobacterium Halobium ◽  
Heavy Atoms ◽  
Isomorphous Replacement ◽  
Atom Localization ◽  
Resolution Structure

We are using electron diffraction to characterize a platinum-containing derivative of bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. This has been undertaken with the dual aims of: 1)using the method of multiple heavy atom isomorphous replacement to obtain high resolution structural information about the protein, and 2)locating heavy atom labelled amino acids in the structure in order to correlate the recently determined sequence with the structural map. A necessary first step in such studies is the location of the heavy atoms in the low resolution structure. This report focusses on ways of dealing with the inherent statistical uncertainties encountered in this heavy atom localization.

Excited-State Proton Transfer in Fluorescent Photoactive Yellow Protein Containing 7-Hydroxycoumarin

2014 ◽  
Vol 896 ◽  
pp. 85-88
Author(s):  
Dian Novitasari ◽  
Hironari Kamikubo ◽  
Yoichi Yamazaki ◽  
Mariko Yamaguchi ◽  
Mikio Kataoka
Keyword(s):  
Hydrogen Bonding ◽  
Excited State ◽  
Proton Transfer ◽  
Fluorescent Protein ◽  
Stokes Shift ◽  
Spectroscopic Studies ◽  
Photoactive Yellow Protein ◽  
Excited State Proton Transfer ◽  
Hydrogen Bonding Network ◽  
Green Fluorescent

Green fluorescent protein (GFP) has been used as an effective tool in various biological fields. The large Stokes shift resulting from an excited-state proton transfer (ESPT) is the basis for the application of GFP in such techniques as ratiometric GFP biosensors. The chromophore of GFP is known to be involved in a hydrogen-bonding network. Previous X-ray crystallographic and FTIR studies suggest that a proton wire along the hydrogen-bonding network plays a role in the ESPT. In order to examine the relationship between the ESPT and hydrogen-bonding network within proteins, we prepared an artificial fluorescent protein using a light-sensor protein, photoactive yellow protein (PYP). The native chromophore of p-coumaric acid (pCA) of PYP undergoes trans-cis isomerization after absorbing a photon, which triggers proton transfers within the hydrogen-bonding network comprised of pCA and proximal amino acid residues. Although PYP emits little fluorescence, we succeeded to reconstitute an artificial fluorescent PYP (PYP-coumarin) by substituting the pCA with its trans-lock analog 7-hydroxycoumarin. Spectroscopic studies with PYP-coumarin revealed that the chromophore takes an anionic form at neutral pH, but is protonated by lowering pH. Both the protonated and deprotonated forms of PYP-coumarin emit intense fluorescence, as compared with the native PYP. In addition, both the deprotonated and protonated forms show identical λmax values in their fluorescence spectra, indicating that ESPT occurs in the artificial fluorescent protein.

Steady-state and time-resolved spectroscopic studies of green-to-red photoconversion of fluorescent protein Dendra2

2014 ◽  
Vol 280 ◽  
pp. 5-13 ◽  
Author(s):  
Nikolay S. Makarov ◽  
Claudiu Cirloganu ◽  
Joseph W. Perry ◽  
Konstantin A. Lukyanov ◽  
Kyril M. Solntsev
Keyword(s):  
Steady State ◽  
Fluorescent Protein ◽  
Spectroscopic Studies ◽  
Time Resolved

scholarly journals Key interactions with deazariboflavin cofactor for light-driven energy transfer in Xenopus (6–4) photolyase

2021 ◽  
Author(s):  
Ayaka Morimoto ◽  
Yuhei Hosokawa ◽  
Hiromu Miyamoto ◽  
Rajiv Kumar Verma ◽  
Shigenori Iwai ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Light Harvesting ◽  
Spectroscopic Studies ◽  
Efficient Energy Transfer ◽  
Time Resolved ◽  
Catalytic Center ◽  
Efficient Energy ◽  
Coulombic Interactions ◽  
Hydrophobic Amino Acid Residue

AbstractPhotolyases are flavoenzymes responsible for light-driven repair of carcinogenic crosslinks formed in DNA by UV exposure. They possess two non-covalently bound chromophores: flavin adenine dinucleotide (FAD) as a catalytic center and an auxiliary antenna chromophore that harvests photons and transfers solar energy to the catalytic center. Although the energy transfer reaction has been characterized by time-resolved spectroscopy, it is strikingly important to understand how well natural biological systems organize the chromophores for the efficient energy transfer. Here, we comprehensively characterized the binding of 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) to Xenopus (6–4) photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction. Mutational analyses of the protein combined with modification of the chromophore suggested that Coulombic interactions between positively charged residues in the protein and the phenoxide moiety in 8-HDF play a key role in accommodation of 8-HDF in the proper direction. This study provides a clear evidence that Xenopus (6–4) photolyase can utilize 8-HDF as the light-harvesting chromophore. The obtained new insights into binding of the natural antenna molecule will be helpful for the development of artificial light-harvesting chromophores and future characterization of the energy transfer in (6–4) photolyase by spectroscopic studies.

Electron Channeling Patterns

1977 ◽  
Vol 35 ◽  
pp. 12-13
Author(s):  
David C. Joy
Keyword(s):  
Electron Diffraction ◽  
Incidence Angle ◽  
Photographic Plate ◽  
Diffraction Effect ◽  
Angle Of Incidence ◽  
Bulk Single Crystal ◽  
Time Resolved ◽  
Electron Channeling ◽  
Spatially Resolved ◽  
Large Bulk

Electron channeling patterns (ECP) were first found by Coates (1967) while observing a large bulk, single crystal of silicon in a scanning electron microscope. The geometric pattern visible was shown to be produced as a result of the changes in the angle of incidence, between the beam and the specimen surface normal, which occur when the sample is examined at low magnification (Booker, Shaw, Whelan and Hirsch 1967).A conventional electron diffraction pattern consists of an angularly resolved intensity distribution in space which may be directly viewed on a fluorescent screen or recorded on a photographic plate. An ECP, on the other hand, is produced as the result of changes in the signal collected by a suitable electron detector as the incidence angle is varied. If an integrating detector is used, or if the beam traverses the surface at a fixed angle, then no channeling contrast will be observed. The ECP is thus a time resolved electron diffraction effect. It can therefore be related to spatially resolved diffraction phenomena by an application of the concepts of reciprocity (Cowley 1969).

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