Sub‐Picosecond Mid‐Infrared Spectroscopy of Phytochrome Agp1 from <i>Agrobacterium tumefaciens</i>

Schümann, Christian; Gross, Ruth T.; Michael, Norbert; Lamparter, Tilman; Diller, Rolf

doi:10.1002/cphc.200700210

Cited by 59 publications

(100 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The excitedstate decay times (43 and 170 ps) agree well with those observed in time-resolved fluorescence experiments (Fig. S4) and fall within the range of time constants observed in other Bph and Cph1 (9,10,12,19). The Lumi-R quantum yield is estimated at 0.13 ( Fig.…”

Section: Photochemistry Of P3: H/d Exchange Effects Reveal Proton-trasupporting

confidence: 84%

“…Recent studies have indicated that 15Za to 15Ea isomerization of the chromophore at the C15═C16 double bond, which causes a flip of pyrrole ring D, accompanies formation of the Pfr state (6). The primary photoproduct, denoted Lumi-R, is formed on the 10-to 100-ps time scale and adopts the 15Ea configuration (8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

“…What is the role of specific chromophore-protein interactions? Why is the quantum yield of Pfr and Lumi-R formation so low at 0.1-0.2 (10,12,17,18)? More generally, what processes determine the fluorescence quantum yield in Bph?…”

mentioning

confidence: 99%

See 2 more Smart Citations

Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome

Toh

Stojković²,

Stokkum³

et al. 2010

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

143

297

View full text Add to dashboard Cite

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15═C16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Here we report that an unusual Bph, RpBphP3 from Rhodopseudomonas palustris, denoted P3, is fluorescent. This Bph modulates synthesis of light-harvesting complex in combination with a second Bph exhibiting classical photochemistry, RpBphP2, denoted P2. We identify the factors that determine the fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. The excited-state lifetime of the biliverdin chromophore in P3 was significantly longer at 330-500 ps than in P2 and other classical phytochromes and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the excited state of biliverdin by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein. This work provides a basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.hydrogen bonds | near-IR fluorescent protein | reaction mechanism | kinetic isotope effect | biophotonics

show abstract

Section: Photochemistry Of P3: H/d Exchange Effects Reveal Proton-trasupporting

confidence: 84%

mentioning

confidence: 99%

See 1 more Smart Citation

Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome

Toh

Stojković²,

Stokkum³

et al. 2010

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

143

297

View full text Add to dashboard Cite

show abstract

“…Absorption of a photon by the redabsorbing state (Pr) leads to the excited Pr* state with a strained C15ϭC16 bond. Isomerization about this bond leads to the second excited state, Lumi-R*, followed by relaxation to form the first photoproduct, Lumi-R (5,6). A proton is then transiently released, possibly from BV and possibly to the tightly bound pyrrole water or the main chain carbonyl oxygen of an adjacent residue, to form Meta-R c .…”

mentioning

confidence: 99%

Structure-guided Engineering Enhances a Phytochrome-based Infrared Fluorescent Protein

Auldridge

Satyshur

Anstrom

et al. 2012

Journal of Biological Chemistry

118

243

View full text Add to dashboard Cite

Background: Engineered variants of the phytochrome photoreceptor are infrared fluorescent proteins. Results: Based on crystal structures, side chain substitutions near the chromophore were combined with monomerization of truncated phytochrome to yield an enhanced fluorophore. Conclusion: Amino acid changes that increase fluorescence discourage photoproduct formation. Significance: This improved infrared phytofluor provides long-wavelength excitation for high signal to noise in tissue and whole animals.

show abstract

“…The P r to P fr photoconversion is initiated by a rapid Z/E isomerization of the C-D methine bridge of the bilin chromophore (19 -22) leading within picosecondsto the formation of the Lumi-R intermediate (23,24). The following thermal relaxations via Meta-R A and Meta-R C intermediates to P fr proceed on the time scale of microseconds and milliseconds (25)(26)(27)(28).…”

mentioning

confidence: 99%

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Borucki

Lamparter

2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to P fr , the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R C and P fr . In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R C substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.

show abstract

Sub‐Picosecond Mid‐Infrared Spectroscopy of Phytochrome Agp1 from Agrobacterium tumefaciens

Cited by 59 publications

References 34 publications

Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome

Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome

Structure-guided Engineering Enhances a Phytochrome-based Infrared Fluorescent Protein

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Contact Info

Product

Resources

About