Photochrmisrry and Phorobioloyy, 1975. Vol. 22, pp. 3-6.

Pergamon Press. Printed in Great Britain

SINGLET ENERGY TRANSFER BETWEEN AROMATIC AMINO ACIDS AND NUCLEIC ACID BASES. THEORETICAL CALCULATIONS T. MONTENAY-GARESTER Museum National d’Histoire Naturelle, Laboratoire de Biophysique, 61, Rue Buffon, 75005 Paris. France (Received 7 October 1974; accepted 11 March 1975)

Abstract4ritical Forster distances for excitation energy transfer at the singlet level between the tyrosyl

or tryptophyl residues of proteins and nucleic acid bases have been calculated. Efficient singlet ener y transfer can be expected from tyrosine to nucleic acid bases both at room temperature (10 < R, < 17 ) and at low temperature (20 < Ro < 23 A). At low temperature nucleic acid bases should be able to

sk

transfer their singlet excitation energy to the indole ring of tryptophan with a reasonable probability (9 < R o < 15 A).

mixed aggregates (Montenay-Garestier, 1974). The present report deals with an evaluation of critical Forster distances for singlet excitation energy transfer between purine or pyrimidine rings and indole or phenol rings at room (fluid medium) and low (rigid medium) temperatures.

INTRODUCTION

The photochemical behavior of nucleic acid-protein complexes can be expected to be different from that of the isolated macromolecules if electronic interactions and/or energy transfer processes can occur in these systems. Efficient energy transfer processes have been demonstrated in nucleic acids (for a review see Eisinger and Lamola, 1970) and in proteins of polypeptides, (Steinberg, 1971; Longworth, 1971). Complex formation between the aromatic rings of tryptophan or tyrosine and nucleic acid bases have been previously demonstrated (Montenay-Garestier and Helene, 1968, 1971 ; Helene et al., 1971). Electron donor-acceptor interactions are involved in these complexes. The rapid freezing of an equimolar mixture of an aromatic amino acid and a nucleoside induces the formation of mixed aggregates whose emission properties are different from those of aggregates of the separated components (Montenay-Garestier and Heline, 1973). Charge-transfer complexes are formed where the indole or phenol ring is the electron donor and the purine or pyrimidine ring acts as the electron acceptor. The fluorescence of each parent molecule is partially or completely quenched and the charge-transfer complex generally exhibits a broad red-shifted emission with a relatively low quantum yield. At the singlet level, this charge-transfer complex behaves as a trap for singlet excitation energy migrating in aggregates (Montenay-Garestier and Heline, 1971; Montenay-Garestier, 1974). It has been recently demonstrated that triplet-triplet energy transfer can take place from bases to tryptophan in mixed aggregates at low (Trp)/(base) ratios and in oligopeptide-poly(A)(or DNA) complexes at low (oligopeptide)/(phosphate)ratios (Helene, 1973). Experimental evidence has also been provided for energy migration from tyrosine to nucleic acid bases at the triplet level as well as at the singlet level in

MATERIALS AND METHODS

Nucleosides and aromatic amino acids were purchased from Calbiochem and used without further purification. At room temperature fluorescence spectra of lo-’ M aq solutions of tyrosine and tryptophan have been recorded on a differential absolute spectrofluorimeter FICA 55 with water as reference. Diluted solutions were used to prevent the distortion of the spectra due to self-absorption effects. Excitation wavelength was 280 nm. At low temperature (77 K), fluorescence spectra of M solutions of tyrosine or tryptophan in a waterpropylene-glycol mixture ( l : l , v/v) were recorded on a JOBIN-YVON spectrofluorimeter corrected for the wavelength dependence of monochromator transmission and of photomultiplier response. Absorption spectra in aqueous and ethanolic solutions at room and at low temperature were recorded on a Cary model 15 spectrophotometer. Fluorescence and absorption spectra recorded in this work do not differ significantly from those previously published (see for example, Eisinger et al., 1969; Eisinger and Lamola, 1971). RESULTS AND DISCUSSION

The Forster theory (1965) for singlet energy transfer after relaxation predicts a transfer rate between two chromophores (donor D and acceptor A ) given by

i )the molar decadic extinction coefficient where ~ ( is on a wavenumber scale (i),,f(?) the fluorescence quantum spectrum normalized to unity, n the index of

3

4

T. MONTENAY-GARESTIER

refraction of the medium intervening between D and A, 7 ithe intrinsic fluorescence lifetime (in the absence of radiationless processes) and R the distance separating the transition dipoles of donor and acceptor molecules. ti is a numerical factor accounting for mutual orientation of the two transition dipoles. For sufficiently fast Brownian rotation of both molecules as compared to the deactivation rate of donor, a value of 2 = 4 may be used. A discussion of appropriate values of 2 that depend on the relative expected orientation of the donor and the acceptor can be found in recent publications (Eisinger and Dale, 1974; Dale and Eisinger, 1974). All the results given below have been calculated using = 2/3. The critical distance R , is defined as the distance at which the rate of energy transfer from the donor D is equal to the sum of rates of all other modes of deactivation R; = 8.8

10-25~2.n-4.4,.~

where J is the previously defined overlap integral, (.I = 1;’ f(i).~($)(di/IV~), and 4Dis the donor kmission quantum yield in the absence of acceptor molecules. In this estimation of R,, several conditions are assumed: (1) The interaction between donor and acceptor is small compared with the width of the unresolved absorption band of the acceptor (the “very weak coupling” case of Forster theory) (Forster, 1965). (2) The dipole approximation is valid. The donoracceptor distances are large compared with the intermolecular distances so that higher multipoles do not need to be taken into account and exchange interactions are negligible. (3) Vibrational relaxation to the lowest excited state of the donor is fast compared with the energy transfer rate (after-relaxation transfer: GuCron et al., 1967). However, in the case of tryptophan it is known that solvent reorientation may take place in the excited state which leads to a broadened, red-shifted fluorescence spectrum in water at room temperature; therefore, energy transfer probabilities have been calculated before and after solvent relaxation (see Tables 1A and 1B). The index of refraction is taken equal to 1.333 at room temperature for aqueous solutions and 1.359 for ethanol glasses at liquid N, temperature. The values of tryptophan and tyrosine fluorescence quantum yields are still the subject of discussions (for a review, see Longworth, 1971). For room temperature measurements two sets of different values were used (Teale and Weber, 1957; Chen, 1967). At liquid N2 temperature mean values (Longworth, 1971) were used. Eiiergy transfer from tryptophan and tyrosine to nucleic w i d bases

Forster critical distances for energy transfer from tryptophan and tyrosine to nucleosides are given in Tables 1A and 1B at room temperature and in Tables 3 and 4 at 77K.

Table 1A. Critical Forster distances for energy transfer from tryptophan fo nucleosides in water at room temperature after relaxation. The overlap integral is negligible in the case of adenosine Ro

~(10-31

Nucleosides Guanosine Cytidine Thymidine

M-’m6)

(4

Ro (A) (4,, = 0.20)t

5.1 4.8 4.3

5.5 5.1

($D = 0.13)+

7.5 5 2.5

4.6

‘Chen (1967). tTeale and Weber (1957).

Table 1B. Transfer distances for energy transfer from tryptophan to nucleosides before relaxation (see text). The overlap integral was calculated using the fluorescence spectrum of tryptophan at low temperature (see text)

R,

(4

Nucleosides

J(10-29 M-’ m”)

(4D = o.20)

Guanosine Cytidine Thymidine

3.4 2.4 1.4

10.3 9.8 9

Table 2. Critical Forster distances for energy transfer from tyrosine to nucleosides in water at room temperature Nucleosides

J(10-28 M-’ m“)

Guanosine Cytidine Thymidine Uridine Adenosine

5.7 4.9 4.75 1.2 0.45

R, (A)

Ro (A)

15.7 15.3 14.6 12.1 10.3

16.8 16.3 15.3 12.9 10.9

(4D= 0.14)’ (4D= O.2l)t

‘Chen (1967). TTeale and Weber (1957).

Table 3. Forster critical distances for singlet energy transfer from tyrosine to nucleosides in ethanolic solutions a t 77 K

Guanosine Cytidine Thymidine Uridine Adenosine

2.3 1.7 1.3 0.95 0.9

23.6 22.2 21.2 20.1 20

dD of tyrosine is taken equal to 0.425 (Longworth, 1971).

Table 4. Critical Forster distances for energy transfer from tryptophan to nucleosides at low temperature (77 K). Fluorescence quantum yield is taken as 0.68 (Longworth, 1971) Nucleosides Guanosine Cytidine Thymidine

J( 10- 2 9 M - m6) 4.5 1.37 0.375

Ro

(4

13.75 11.4 9.2

Energy transfer between amino acids and DNA bases

5'

In Table lB, we evaluated the critical distances for Table 5'. Forster critical distances for singlet energy energy transfer from tryptophan to nucleic acid bases transfer @om nucleosides to tyrosine at 77 K. The fluorescbefore solvent relaxation using the fluoresence spec- ence quantum yields used for nucleosides are those given by Eisinger and Lamola (1971) for nucleotides trum of tryptophan at low temperature in an ethanolic glass to calculate the overlap integral J. Several Nucleosides & R, (A) J(10-29M-' m6) cases must be distinguished. 0.045 0.13 4.65 (i) If an isolated tryptophan molecule transfers its Guanosine Cytidine 1.62 0.05 8.5 excitation energy to a nucleic acid base in water at Thymidine 1.62 0.16 8.8 room temperature before relaxation, one must use the Adenosine 2.75 0.01 6 lifetime of the non-relaxed state to evaluate R,. This lifetime is not known experimentally. One may use Table 6. Critical Forster distances for energy transfer from the rotational correlation time for H,O molecules, nucleosides to tryptophan at low temperature (77 K). The which should be of the order of lo-" s. The relaxed fluorescence quantum yields used are those of nucleotides given by Eisinger and Lamola (1971) state of tryptophan has a lifetime of about 4 x lo-' 8. As seen in Table lB, the overlap integral is about Nucleosides J(10-28M-' m6) & Ro (A) two orders of magnitude larger before relaxation than after relaxation. The lifetime of the non-relaxed state Guanosine 1.25 0.13 12.5 5.95 0.05 13.9 (and therefore its quantum yield $D) should be two Cytidine 1.71 0.16 15.2 orders of magnitude smaller than that of the relaxed Thymidine 3.15 0.01 9.5 state. Therefore, the critical distances Ro correspond- Adenosine ing to this case are quite similar to those given in a factor of one-half of the transfer distances from tyroTable 1A. (ii) If one is dealing with a protein-nucleic acid sine to bases, the transfer from tyrosine to nucleic complex in which a tryptophan residue is not able acid bases is expected to be much more efficient than to relax in the excited state because, e.g. this residue the transfer in the opposite direction, since the is not accessible to solvent molecules, then one must transfer probability increases as the sixth power of use the actual lifetime and quantum yield of this resi- the critical distance. If Ro is the critical Forster disdue to calculate R,. This quantum yield will, of tance calculated for the energy transfer from a donor course, depend on the particular case under investiga- D to an acceptor A and R& the critical Forster distion. In Table lB, R, values have been calculated tance from the acceptor A to the donor D, the true using a 4Dvalue of 0.2. It can be seen that the energy critical Forster distance R from donor D to acceptor transfer from tryptophan to bases will be more prob- A, taking back-transfer into account, is given by able than after relaxation. Rb = Rg - Rb6(1 + 2 ~ ) (iii) In most proteins, the environment of trypto- where CI is the ratio of the intensities absorbed by phan is such that partial relaxation of the excited species A and D, respectively, at the exciting wavestate is possible. Therefore, the emission spectrum will length (Montenay-Garestier and Helene, to be pubbe intermediate between those calculated using the lished). fully relaxed emission spectrum at room temperature In Table 6 are given3he critical distances R, for and the non-relaxed emission spectrum observed in energy transfer from nucleosides to tryptophan at low rigid medium at low temperature. Thus, R , values will temperature. Comparison of the critical distances calbe intermediate between those given in Table 1A and 1B. culated in both directions (Tables 4 and 6) leads to At low temperature, the overlap between the fluor- the conclusion that transfer is expected to occur from escence spectrum of tyrosine and the absorption spec- nucleosides to tryptophan except in the case of tra of nucleosides is more important than at room guanosine (although the fluorescence quantum yield temperature. Forster distances are expected to in- of tryptophan at low temperature is high). Transfer crease accordingly (see Tables 2-3). Values for critical probabilities appear to be less than in systems involvenergy transfer distances from tyrosine to nucleosides ing tyro sine. at low temperature (Table 3) are higher than correTransfer probabilities calculated here might be of sponding transfer rates between tyrosine and trypto- importance in the photochemistry of nucleic acidphan previously calculated by Eisinger (1971) at 77 K. protein systems. The aromatic rings of tryptophan and tyrosine have been shown to complex. Even withEnergy transfer @om nucleic acid bases to tryptophan out complex formation, the eventuality of long-range or tyrosine energy transfer mechanisms should be taken into Due to the low quantum yield (10-4-10-5) of nu- account in the photochemical behavior of nucleic cleosides at room temperature (Vigny, 1974), the criti- acid-protein complexes. Studies are in progress in our cal distances for energy transfer from bases to tyro- laboratory to provide experimental evidence for such sine or tryptophan will be negligible. At low tempera- energy transfer processes. ture calculations relative to the transfer from bases Acknowledgements4 wish to thank Prof. G. Laustriat to tyrosine lead to non-negligible values of critical (Strasbourg) and Dr. C . Helbne (Orleans) for valuable critidistances (Table 4). Though these distances are within cisms and helpful discussions.

6

T. MONTENAY-GARESTIER REFERENCES

Chen, R. F. (1967) Anal. Letters 1, 35-42. Dale, R. E., and J. Eisinger (1974) Biopolymers 13, 1573-1605. Eisinger J., and R. E. Dale (1974) J . Mol. Biol. 84, 643-647. Eisinger J., and A. A. Lamola (1971) In Excited States of Proteins and Nucleic Acids (Edited by R. F. Steiner and I. Weinryb). pp. 107-198. Plenum Press, New York. Eisinger J., B. Feuer and A. A. Lamola (1969) Biochemistry 8, 3908-3915. Forster Th. (1965) In Modern Quantum Chemistry (Edited by 0. Sinan6glu). pp. 93-137. Academic Press, New York and London. Gueron M., J. Eisinger and R. G. Shulman (1967) J. Chem. Phys. 47, 4077-4091. HtlBne C . (1973) Photochem. Photobiol 18, 255-262. HClkne C., T. Montenay-Garestier and J. L. Dimicoli (1971) Biochim. Biophys. Acta 254, 349-365. Longworth, J. W. (1971) In Excited States of Proteins and Nucleic Acids (Edited by R. F. Steiner and I. Weinryb), pp. 319484. Plenum Press, New York. Montenay-Garestier, T. (1975) International Conference on the Excifed States of Biological Molecules (Edited by J. B. Birks), Lisbon. Wiley, New York (in press). Montenay-Garestier, T., and C . Helene (1968) Nature 217, 844-845. Montenay-Garestier, T., and C . Helkne (1971) Biochemistry 10, 3W306. Montenay-Garestier, T., and C. Helene (1973) J. Chem. Phys. 70, 1385-1390. Steinberg, I. Z. (1971) Ann. Rev. Biochem. 40, 83-1 14. Teak, F. W. J., and G. Weber (1957) Biochem. J. 65, 47M82. Vigny, P. (1974) Thtse de Doctorat d’Etat (Paris).

Singlet energy transfer between aromatic amino acids and nucleic acid bases. Theoretical calculations.

Photochrmisrry and Phorobioloyy, 1975. Vol. 22, pp. 3-6. Pergamon Press. Printed in Great Britain SINGLET ENERGY TRANSFER BETWEEN AROMATIC AMINO ACI...
333KB Sizes 0 Downloads 0 Views