J . Phys. Chem. 1986, 90,4578-4580
4578
These experimental features of phenanthrylammonium ion18-crown-6 complexes are very similar to those of naphthylammonium ion-1 8-crown-6.14
Concluding Remarks Complex formation of phenanthrylammonium ions with 18crown-6 decreases significantly the proton dissociation rate k l in the excited singlet state, resulting in an increase of its lifetime or fluorescence quantum yield. For 2- and 3-RN+H3-crown complexes, they are too stable to dissociate into the neutral amine species plus proton in the excited state. The hydrogen-bonded exciplex (RNH2-crown)* is produced by deprotonation of (RN+H3-crown)*for 1-, 4-, and 9RN+H,-crown complexes. The
excited-state proton-traqsfer reaction is a one-way process since the back protonation rate.&, is negligibly small compared to those of the other competitive decay processes. There is a large steric effect on protonation of the amino group of the excited neutral complex. Thus, there is no excited-state prototropic equilibrium in the RN+H3-crown complexes. The Corey-Pauling-Kolton molecular model proposed by Izatt et al.25is strongly supported by the present work. Registry No. 1R'NH,, 103477-68-5;2R+NH3,103498-76-6;3R+NH,, 103477-69-6; 4R+NH3, 103477-70-9; 9R+NH3, 103477-71-0; 1 RNHZ, 4176-53-8; 2RNH2, 3366-65-2;3RNH2, 1892-54-2;4RNH2, 17423-48-2; 9RNH2, 947-73-9; 18-crown-6, 17455-13-9.
Control of Bilin Transition Dipole Moment Direction by Macromolecular Assembly: Energy Transfer in Allophycocyanin Sheila W. Yeh, Department of Chemistry and the Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720
Alexander N. Glazer,* Department of Microbiology and Immunology, University of California, Berkeley, California 94720
and John H. Clark Amoco Research Center, Amoco Corporation, Naperville, Illinois 60566 (Received: February 5, 1986)
The monomer a@ of the photosynthetic accessory protein allophycocyanin carri@&wne covalently bound phycocyanobilin on each subunit. The emission decay of the monomer is made up of two c o m p o n w of similar amplitudes of 0.7 and 1.9 ns. The wavelength-independentvalue of 0.40 i 0.02 of the emission polarization of ab shows that the absorption and emission transition dipole moments of each of the two bilins are parallel to within 23 f 2'. The trimer, (a&, exhibits a single emission 0.08 i 0.03, is independent of excitation decay component of lifetime 1.76 ns. The value of the emission polarization of wavelength. From this value, the angle between the absorption and emission transition dipole moments of the chromophores reflects the of the allophycocyanin trimer is 50 f 3'. The difference in the emission polarization between cub and control of bilin transition dipole moment orientation by the specific assembly of monomers into trimers.
Introduction Cyanobacterial and red algal phycobiliproteins are macromolecular building blocks of the phycobilisome, a particle optimized for light energy absorption and highly directional energy transfer.'.2 Allophycocyanin is the simplest of the these proteins with respect to bilin content. The monomer, ap, carries a single phycocyanobilin on each of its two polypeptide chain^.^ Absorption and circular dichroism measurements on the monomer and on the isolated cy and p subunits indicate that in the monomer the two chromophores are independent of each ~ t h e r . ~Upon . ~ assembly into a disk-shaped trimeric aggregate, the spectroscopic properties of allophycocyanin undergo substantial changes. The absorption maximum, which lies at 620 nm in the monomer, shifts to 650 nm in the trimer and exhibits a shoulder at 620 nm.6-8 ( 1 ) Glazer, A. N. Biochim. Biophys. Acta 1984, 768, 29. (2) Glazer, A. N. Annu. Rev. Biophys. Biophys. Chem. 1985, 14, (3) Glazer, A. N.; Fang, S . J . Biol. Chem. 1973, 248, 659.
47.
(4) Csatorday, K.; MacColl, R.; Csizmadia, V.; Grabowski, J.; Bagyinka, C. Biochemistry 1984, 23, 6466. (5) Cohen-Bazire, G.; BBguin, S . ; Rimon, S . ; Glazer, A. N.; Brown, D. M. Arch. Microbiol. 1977, 11 I , 225. (6) Gysi, J. R.; Zuber, H. Biochem. J . 1979, 181, 577. (7) Murakami, A.; Mimuro, M.; Ohki,K.; Fujita, Y. J . Biochem. 1981, 89, 79. ( 8 ) Mimuro, M.; Murakami, A.; Fujita, Y. Arch. Biochem. Biophys. 1982, 215. 266.
0022-3654/86/2090-4578$01.50/0
Whereas the circular dichroism (CD) spectrum of the monomer corresponds to the absorption spectrum and shows a single positive Cotton effect centered at 620 nm with a shoulder at 586 nm? the trimer shows positive C D bands at 632 and 656 nm.4.9 The monomer fluorescence emission maximum lies at 645 nm,l0 that of the trimer at 660 nm1.5311These differences in spectroscopic properties are clearly the consequence of the new intersubunit contacts and/or conformational changes attendant upon the assembly of the trimer. Since the directionality of energy transfer in phycobiliproteins is observed only in higher aggregates such as the allophycocyanin trimer, an analysis of the meaning of the differences in properties that accompany the assembly of such aggregates is a prerequisite to the understanding of the interplay between macromolecular structure and energy-transfer function. The changes in spectroscopic properties associated with trimer formation have been interpreted4," to arise from exciton interaction between bilins on a and p subunits of neighboring monomers in the trimer (Figure 1). This inference is based largely on the appearance of the C D spectrum of the trimeric aggregate. In this report, we describe the steady-state absorption and fluorescence polarization and the time-resolved fluorescence emission of the allophycocyanin monomer and trimer. These (9) Lundell, D. J.; Glazer, A. N. J . Biol. Chem. 1983, 258, 8708. (IO) Ong, L. J.; Glazer, A. N. Physiol. Veg. 1985, 23, 777. ( 1 1 ) Canaani, 0. D.; Gantt, E. Biochemistry 1980, 19, 2950.
0 1986 American Chemical Society
The Journal of Physical Chemistry, Vol. 90, No. 19, 1986 4579
Energy Transfer in Allophycoyanin A
Figure 1. Diagram of the assembly of allophycocyanin monomers into trimers. The areas of greatest interaction between subunits within individual monomeric aggregates are stippled. I
"
"
'
"
1
h 0
2
4
6
8
Nanoseconds
OJ
Figure 3. Plots of emision intensity (I) as a function of time from A. uariabilis allophycocyanin aggregates. The top two panels give temporal profiles of emission for trimeric (a@),aggregates. The data (noisy, solid curve) are superimposed with a single-exponentialfit, giving a fluorescence lifetime of 1.76 ns. The semilogarithmic plot (topmost panel)
t 500
550
600 Nanometers
650
700
Figure 2. (A) Absorption spectra of A. variabilis allophycocyanin for two different aggregation states. The dashed curve is the absorption spectrum of monomers, and the solid curve is of the trimeric state. (B) Steady-state polarization spectra of A. uariabilis allophycocyanin. The dotted curve gives the polarization spectrum of monomers, while the solid curve is the polarization spectrum of trimers. studies permit the assignment of the relative orientations of the absorption and emission transition dipole moments of the bilin chromophores in cy@ and in (cy@),. The results show that, in going from cy@ to (a& there is a large change in the inclination of the transition dipole moment for fluorescence emission relative to the direction of the absorption dipole moment.
Experimental Methods Allophycocyanin was purified from Anabaena uariabilis according to standard procedures.12 The solutions used for all steady-state spectroscopic studies were of low optical density (A650 5 0 . 1 A/cm). High salt buffers (0.75-0.80 M phosphate, pH 7.0) ensured the stability of the trimeric aggregation state even at such low protein concentrations. Monomer formation was induced by the use of the chaotropic salt NH4SCN (0.6 M NH,SCN, 0.05 M phosphate, pH 7.0). Steady-state emission spectra and emission polarization spectra were measured with a computer-controlled Spex Fluorolog 11. The value, P, of the fluorescence polarization was calculated by conventional procedures." Emission from the samples was timeresolved by using an ultrafast streak camera.14 An optical (1 2) Bryant, D. A.; Glazer, A. N.; Eiserling, F. A. Arch. Microbiol. 1976, 110, 61. (13) Chen, R. F.; Bowman, R. L. Science 1965, 147, 129.
(14) Webb, S. P.; Yeh, S. W.; Clark, J. H. Reu. Sci. Insrrum., manuscript in preparation.
indicates the quality of the single-exponential fit. The lower panels are signals that have been time-resolved from monomeric allophycocyanin aggregates, (a@).In the lowest of the four panels, the signal is shown with two fits superimposed. The dashed curve is a single-exponential fit while the solid, smooth curve is a double-exponential fit to the data. The superiority of the double-exponential fit is clearly seen in the semilogarithmic plot shown in the third panel. The parameters of the doubleexponential fit are a , = 1.0, r , = 0.7 ns; a2 = 1.6, T~ = 1.9 ns. parametric source gave tunable excitation pulses.15 Fluorescence was filtered through a long wavelength pass filter (Corning 2-58).
Results and Discussion The absorption spectrum of the allophycocyanin monomer is shown in Figure 2A. The shape of this spectrum is the same as the shapes of the absorption spectra of each of the isolated cy and p subunit^.^ It is also observed that the emission spectrum of the monomer (Figure 2B) has the same shape as the emission spectra of the individual subunit^.^ Consequently, in the monomer, the two bilins do not have distinctive steady-state spectroscopic signatures. However, as shown in Figure 3, the time-resolved emission from cy@ consists of two distinct components of comparable magnitude with lifetimes of 0.7 and 1.9 ns. Furthermore, the steady-state polarization of the monomer emission is independent of excitation wavelength (Figure 2B). The observed value of 0.40 f 0.02 for the monomer emission polarization predicts that the absorption and emission transition dipole moments of each of the two bilin chromophores are parallel to within 23 f 2'. The pronounced change in the absorption spectrum accompanying trimer formation is seen in Figure 2A. The trimer emission rises sharply at the red edge of the absorption band and is significantly shifted to longer wavelengths relative to that of the monomer. In contrast to the observed biexponential decay of the monomer emission, the time-resolved trimer emission can be accurately fitted with a single exponential giving a lifetime of 1.76 ns. The fact that there is only a single lifetime for trimer emission decay can be accounted for by either of two explanations: (a) (15) Nathel, H.; Guthals, D. M.; Anthon, D. W.; Clark, J. H. J . Opr. SOC. Am. 1983, 73, 1897. Nathel, H.; Guthals, D. M.; Anthon, D. W.; Clark, J. H. Op?. Lerf., to be submitted for publication.
4580 The Journal of Physical Chemistry, Vol. 90, No. 19, 1986 trimer assembly is accompanied by the formation of bilin exciton pairs as diagrammed in Figure 1; or (b) that no exciton is formed, but extremely rapid transfer of energy takes place between the same bilin chromophores with one acting as an emitter. There is considerable precedent for the latter interpretation. In numerous previous studies of phycobiliproteins that contain chemically different bilins with well-separated absorption maxima, it has been determined that the emission emanates entirely from the longest wavelength absorbing chromophores.16 On the basis of such studies, the donor bilin chromophores that absorb but do not fluoresce were termed “s” (for sensitizing) and the acceptor bilins that both absorb and fluoresce were termed In an attempt to distinguish between the above two explanations, we have examined the steady-state excitation polarization spectrum of the trimer. As shown in Figure 2B, the trimer emission polarization is independent of excitation wavelength. The magnitude of the polarization is 0.08 f 0.03. This corresponds to an angle of 50 f 3O between the direction of the transition dipole moment(s) of the absorbing chromophore(s) and the emitting chromophore. The invariance of this angle with excitation wavelength demonstrates that the absorption transition dipoles of the “s” and “f“ chromophores are parallel. Taken alone, in spite of the improved insight into the relative orientations of the chromophores provided by these measurements, we cannot distinguish between the exciton and the rapid-energy-transfer models. A high-resolution crystal structure of allophycocyanin would enable us to answer this question definitively. However, other recent X-ray crystallographic a n a l y ~ e s l would ~~~* suggest that the rapid-energy-transfer model is correct. The crystal structure of a C-phycocyanin trimer has been determined at 3-A reso1ution.l’ In contrast to allophycocyanin, C-phycocyanin carries three phycocyanobilins per ab monomer. Two of these bilins are linked to cysteinyl residues in positions that correspond to the locations of the bilin-linked cysteinyl residues in allophycocyanin, and the third is attached near the carboxyl terminus of the @-subunitof C-phycocyanin to a cysteinyl residue in an amino acid sequence that has no counterpart in allophycocyanin.’ The sequences of allophycocyanin a and /3 subunits are highly homologous to the sequences of the corresponding C-phycocyanin subunits, and secondary structure predictions indicate similarities in folding in the two sets of polypeptide chains.I9 Electron microscopic studies show that both C(16) Teale, F. W. J.; Dale, R. E. Biochem. J . 1970, 116, 161. (17) Schirmer, T.; Bode, W.; Huber, R.; Sidler, W.; Zuber, H. J. Mol. Biol. 1985, 184, 257. (18) Riimbeli, R.; Schirmer, T.; Bode, W.; Sidler. W.; Zuber, H. J . Mol. Biol. 1985, 186, 197. (19) Fiiglistaller, P.; Suter, F.; Zuber, H. Hoppe-Seyler’s 2. Physiol. Chem. 1983, 364, 691.
Yeh et al. phycocyanin and allophycocyanin trimers are disk-shaped molecules of similar diameters and thicknesses.12*20-21 It is reasonable to infer, therefore, that corresponding bilins, those bound to aCys-84 and /3-Cys-82 in C-phycocyanin and a-Cys-80 and @Cys-8 1 in allophycocyanin, occupy similar positions relative to each other in the two proteins. Schirmer et al.I7 report that the distance between these two bilins in C-phycocyanin is =20 A. If in allophycocyanin trimers the two bilins are indeed separated by =20 8,an exciton model would appear to be ruled out. If the rapid-energy-transfer model is correct, then the polarization data on the trimer permit the calculation of the orientation factor K~ for the Forster-type dipole-dipole energy transfer. The polarization spectrum of (a& is flat except for a small positive deviation at short wavelengths, presumably due to a small contribution from the highly polarized monomer emission (Figure 2). The constancy of the emission dipole moment between excitation of s- or f-type chromophores signifies that the transition dipole moments of the two chromophores making up a single (s,f) pair are parallel. The Forster theory of dipole-dipole energytransfer rates has a strong dependence on the relative directions of the donor and acceptor transition dipole moments,22found in the term K ~ .The value of K~ is a maximum when the two chromophores are parallel, and therefore the energy-transfer rate between the two allophycocyanin chromophores is the fastest that would be predicted by the Forster theory. For two chromophores that are separated by 20 A, and with parallel transition dipole the transfer rate constant could be as short as a moments, (d), fraction of a picosecond, assuming optimal overlap of donor and acceptor spectra. We have shown recently that energy transfer between the chromophores in allophycocyanin occurs on a time scale of less than 8
Acknowledgment. Work in the Laboratory of Chemical Biodynamics and the Department of Chemistry was supported by the Office of Energy Research, Office of Basic Energy Sciences, Chemical Sciences Division of the Department of Energy, under contract DE-AC03-76SF00098. Work in the Department of Microbiology and Immunology was supported by NSF grant DMB 8518066. S.W.Y. was a Procter and Gamble Fellow. We are grateful to Linda J. Ong for providing the allophycocyanin preparations. (20) Eiserling, F. A.; Glazer, A. N. J. Ultrusrruct. Res. 1974, 47, 16. (21) Morschel, E.; Koller, K.-P.; Wehrmeyer, W. Arch. Microbiol. 1980, 125, 43. (22) Forster, Th. W. “Action of Light and Organic Molecules”. Modern Quantum Chemistry, Pur? II& Sinanoglu, O . , Ed.; Academic: New York, 1965; p 93. (23) Glazer, A. N.; Yeh, S. W.; Webb, S.P.; Clark, J. H.Science 1985, 227, 419.
