OPTICAL SPECTROSCOPIC STUDIES OF PERIDININ AND PERIDININ-CHLOROPHYLL-PROTEIN (PCP) COMPLEXES Nirmalya Chatterjee,1 Dariusz M. Niedzwiedzki,1 Kazuyoshi Aoki,2 Tim Schulte,3 Shigeo Katsumura,2 Eckhard Hofmann,3 and Harry A. Frank1 1Department of Chemistry, University of Connecticut, Storrs, CT, 06269-3060, USA, 2Department of Chemistry, Kwansei Gakuin University, 669-1337, Hyogo, Japan, 3Lehrstuhl für Biophysik, Ruhr-Universität Bochum, 150, D-44801, Bochum, Germany
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PerOlEs
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Figure 3. Transient absorption spectra of peridnin and synthetic derivatives in methanol and n-hexane taken at different delay times.
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Objectives
1 YFYF
Examine the effect of structural changes on the spectral properties of the individual molecules Elucidate the factors controlling the dynamics of the excited states of peridinin Assign the deconvolved spectral bands of peridinin to specific structures in the PCP complex Understand the effect of changes in amino acids on the spectroscopic and energy transfer characteristics of PCP
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HO
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CH3
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Peridinin (all-trans)
HO OH
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CH3
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H3C
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Normalized absorbance
A PerOlEs
PerAcEs
• HO
O HO
PerAcEs (15,15'-cis)
O O
C
CH3
300
-2 Figure 4. Structure of main-form Peridinin-chlorophyll-protein (PCP) with the positions of the mutated amino acid residues and peridinins indicated on the N-domain side of the PCP monomer. The mutants (given below) were tested against a ‘control’ refolded N-domain PCP (RFPCP) for changes in spectral properties: Glu133Gln (EQ), Tyr108Phe/Tyr136Phe (YFYF), Glu133Lys/Lys124Phe (EKKF), Glu133Lys/Lys124Asn (EKKN). RFPCP in buffer Chl-a Per 1 (479 nm) Per 2 (510 nm) Per 3 (529 nm) Per 4 (548 nm) Fit
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600
Figure 2. Structures and steady-state absorption spectra of peridinin and two synthetic derivatives.
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Peridinin
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MeOH n-hexane
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Figure 1. (left) A. carterae. (Edmund Nash and C. J. Howe, Cambridge University, UK http://www.bio.cam.ac.uk/~howelab/research.htm). (right) Alexandrium fundyense bloom off the coast of Maine. (WHOI, Woods Hole, MA, US - www.whoi.edu/redtide)
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70 fs 2 ps ND
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YFYF
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Figure 6. Wavelength shifts of deconvolved peridinins in the various mutants relative to RFPCP.
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Per 3 (529)
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YFYF EKKF EKKN EQ
Per 1 (479)
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0 48
C
PerAcEs
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-3
MeOH
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n-hexane
Peridinin is a C37 carotenoid found in the Peridinin Chlorophyll-Protein (PCP) from marine dinoflagellates including Amphidinium carterae. An unusual property of peridinin is that its lowest excited singlet state (S1) lifetime depends strongly on solvent polarity, e.g. the S1 lifetime is 161 ps in n-hexane and 12 ps in methanol. It remains to be elucidated whether a physiological role exists for this behavior. Steady-state and ultrafast time-resolved optical spectroscopic studies were done on purified peridinin, two synthetic peridinin derivatives, and recombinant wild type and mutant N-domain PCPs refolded in the presence of peridinin and chlorophyll-a. The investigation of the isolated molecules provide information as to what structural features are responsible for the spectroscopic properties of peridinin. Spectral deconvolution of the steady-state absorption spectra of the refolded PCP complexes reveal the band profiles of the underlying peridinins. Ultrafast time-resolved experiments on the proteins show subtle differences between the complexes suggestive of the robust nature of the pigment-protein complex architecture.
Peridinin
-2
Abstract
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Figure 8. (above) Evolution-associated difference spectra (EADS) obtained from global fitting of transient spectra of RFPCP and the double-mutant YFYF.
Figure 7. (right) Representative transient spectra of RFPCP and the double-mutant YFYF in 60% glycerol-buffer at room temperature.
Conclusions
The lactone ring in peridinin is the primary source of the solvent-dependent spectra and dynamics of the molecule. Transitions involving an intramolecular charge transfer state dominate the transient absorption spectra of the molecules in methanol, whereas in n-hexane the transitions originate primarily from the S1 state. Spectral deconvolutions of the various PCP complexes reveal the underlying band structure of the peridinins. Mutations alter the spectra of select peridinins. Global fitting of the transient data from the mutants require an extra EADS component compared to RFPCP for a good fit. The interpretation of these effects is on-going.
Reference
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Wavelength/nm
Figure 5. Spectral deconvolution of the steady-state absorption spectrum of RFPCP into the underlying peridinin and chlorophyll-a spectra. Numbers in parentheses denote the position of the 0-0 vibronic of the peridinin spectrum.
1. Bautista, J. A.; Frank, H. A. et al., J. Phys. Chem. B (1999) 103, 8751-58. 2. Zigmantas, D.; Polívka, T. et al., J. Phys. Chem. B (2003) 107, 5339-48. 3. Hofmann, E.; Welte, W.; et al., Science (1996) 272, 1788-91.
Acknowledgments This work is supported by grants from the National Institutes of Health (GM-30353) and the University of Connecticut Research Foundation.
