Photochemivrry and Photobiology Vol. 56, No. 5 , pp. 725-133, 1992

Printed in Great Britain. All rights reserved

0031-8655/92 $05.00+0.00 Copyright 0 1992 Pergamon Press Ltd

THE DISTANCE BETWEEN THE PHYTOCHROME CHROMOPHORE AND THE N-TERMINAL CHAIN DECREASES DURING PHOTOTRANSFORMATION. A NOVEL FLUORESCENCE ENERGY TRANSFER METHOD USING LABELED ANTIBODY FRAGMENTS DAVIDL. FARRENS~*, MARIE-MICHBLE CORDONNIER~, LEE H. PRATT~ and PILL-SOON SONG’? ‘Department of Chemistry and Institute for Cellular & Molecular Photobiology, University of Nebraska, Lincoln, NE 68588-0304, USA and 2Department of Botany, University of Georgia, Athens, G A 30602, USA (Received 3 February 1992; accepted 11 May 1992) Abstract-A novel antibody-fluorescence method has been developed to elucidate the chromophore topography in phytochrome as it undergoes a photochromic transformation. Forster energy transfer from N-terminal bound, fluorescently labeled Oat-25 Fab antibody fragments to the phytochrome chromophore was measured. The results suggest that the chromophore moves relative to the Nterminus upon the Pr -+ Pfr phototransformation. This conclusion is consistent with previous models which have proposed a reorientation and an interaction of the Pfr chromophore with the N-terminus. The method described appears to be the first study of a Forster energy transfer measurement using a donor-label attached to a Fab fragment of a photosensor protein.

INTRODUCTION

The photosensitive protein phytochrome is found in all higher plants, where it acts as a light-activated morphogenetic switch. Upon irradiation with red light, the inactive form of the protein (Pr)$ is converted to a physiologically active, far-red light absorbing form (Pfr). This reaction is photoreversible and is responsible for plants’ ability to sense changes in their surrounding light conditions (for a most recent review, see Riidiger and Thiimmier, 1991; Thomas and Johnson, 1991). Recently, much of the nature of the chromophore responsible for the phytochrome phototransformation has been elucidated. The chromophore is a linear tetrapyrrole (Lagarias and Rapoport, 1980), similar to the bilin chromophore found in C-phycocyanin (Schirmer et al., 1987). The primary photochemical step in the Pr + Pfr phototransformation is thought to involve a Z -+ E isomerization about the ClrCIh double bond of the chromophore (Riidiger et al., 1983; Thiimmler and Riidiger, 1983; Farrens et al., 1989a; Rospendowski et al., 1989; Fodor et al., 1990; Mizutani et al., 1991). *Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA. ?To whom correspondence should be addressed. $Abbreviations: DMF,N,N-dimethylformamide; EDTA, ethylenediaminetetraacetic acid; Fab, fluorescently labeled antibody; IRF, instrument response function; Pfr, far-red light absorbing form of phytochrome; PMT, photomultiplier tube; Pr, inactive phytochrome; SAR. specific absorbance ratio; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TAC, timeto-amplitude conversion.

The protein environment surrounding the phytochrome chromophore defines the light sensing properties of the protein. Analysis of limited proteolytic digestion (Riidiger, 1987; Yamamoto et al., 1987) as well as peptide deletion-mutants (Deforce et al., 1991) have suggested that certain regions of the protein are involved in the Pr -+ Pfr phototransformation, especially those near the N-terminus (Vierstra et al., 1987; Chai et al., 1987). The chromophore reorients upon Pr to Pfr phototransformation (Ekelund et al., 1985; Tokutomi and Mimuro, 1989). The chromophore also becomes more exposed in the Pfr form to exogenously added chemicals (Hahn et al., 1984; Thiimmler et al., 1985; Farrens et al., 1989b), again suggesting a change or reorientation in the Pfr chromophore-protein relationship. Note that all of the studies mentioned above used only indirect deductions to assume Nterminal-chromophore interaction (i.e. when the Nterminus is removed, the chromophore absorption spectrum changes). Direct evidence for chromophore-N-terminal interaction in the native protein is lacking. The location and orientation of the chromophore in native 124 kDa phytochrome has not been reported. The work presented here addresses this problem. Information about chromophore location and re-orientation in proteins can be obtained using non-radiative energy transfer. Energy transfer, as first described by Forster (1948), can be used as a “spectroscopic ruler’’ to determine distances between two chromophores having overlapping absorption and fluorescence spectra (Stryer and Haughland, 1967).

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et al. DAVIDL. FARRENS

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Unfortunately, n o unique site exists in phyto-

chrome where a single fluorescent label can b e introduced. Therefore, in the present study, we developed a novel method using fluorescently labeled antibody fragments (Fab). T h e antibody used (Oat-25) is known t o bind near the N-terminus of phytochrome (Cordonnier et al., 1985; Pratt et al., 1988). In this paper we report the spatial relationship of the N-terminal t o the phytochrome chromophore as studied by energy transfer from t h e fluorescently labeled F a b bound t o phytochrome in the Pr and Pfr forms. MATERIALS AND METHODS

Materials Phytochrome. Native 124-kDa phytochrome was isolated and purified from etiolated oat seedlings (Avena sativa, Garry oat), according to a modification of the method of Chai et al. (1987). Briefly, in this method phytochrome is extracted from 3.5-day old etiolated shoots and precipitated by ammonium sulfate fractionation. The phytochrome/protein solution obtained is next adsorbed onto a hydroxyapatite column and the column is washed with increasing concentrations of potassium phosphate buffer, pH 7.8. The phytochrome is finally eluted and then precipitated in a final ammonium sulfate fraction/purification step. The following modifications to the method of Chai et al. (1987) were found to increase the yield of phytochrome and decrease the amount of time for isolation: (i) Combining two to three tissue extractions before application to the column. The 20% ethylene glycol concentration in the buffers allows the tissue extracts to be stored for several hours at 258 K, allowing more than one tissue extraction to be performed. (ii) Use of leupeptin (1-10 pglmL). Our results showed that leupeptin strongly inhibited proteases in the plant extracts not affected by serine protease inhibitor phenylmethanesulfonyl fluoride (4 mM). We kept phytochrome to which leupeptin had been added for as long as 3 months at 277 K with no observed proteolytic degradation. (iii) Performing an extra ammonium sulfate fractionation (17 g ammonium sulfate1100 m L protein solution), before applying to the hydroxyapatite column. This additional fractionation step eliminated a large amount of “junk” proteins that can cause column blockage, ( i v ) Use of calcium tartrate gel instead of hydroxyapatite f o r the column purification step. Calcium tartrate gels prepared as described (Akhrem and Drozhdenyuk, 1989) have a higher flow rate than hydroxyapatite. The last two modifications [(iii) and (iv)] allow for a much faster flow rate through the purification column, as reported by Lapko (1989). The SAR values (specific absorbance ratio of the absorbance at 666 nm to the absorbance at 280 nm) of the phytochromes used were in the range of 0.8-0.9 and exhibited a single 124 kDa band on SDS-PAGE. The absorbance measurements were made using a Hewlett-Packard 8452 diode array spectrophotometer. All samples were stored in 20 mM potassium phosphate buffer, pH 7.8, containing 1 mM EDTA and 1 p,g/mL leupeptin. All measurements were taken at 279-281 K. Chemicals. Routine chemicals and solvents were purchased from commercial sources, and deionized water was used for buffer preparations. Calcium tartrate was prepared as described in Akhrem and Drozhdenyuk (1989). When following this procedure, enough 1.5 M phosphate buffer should be added at the gel boiling stage to make the final pH after boiling between 7.2 and 7.6 (V. Lapko, personal communication).

Antibody purification and labeling for Forster energy transfer studies Oat-25 anti-phytochrome antibody purification and Fab generation. Oat-25 mouse monoclonal antibodies were purified from mouse ascites fluid by ammonium sulfate fractionation and Protein-A column chromatography (Harlow and Lane, 1988). Fluorescently labeled antibody fragments from the purified monoclonals were generated using a Pierce ImmunoPure Fab preparation kit (Pierce Chemical Company, Rockford, IL). Fluorescent labeling of Oat-2S Fab antibody fragments. The Fab fragments prepared as above were labeled using a newly synthesized carbocyanine dye, kindly provided by Dr. A. Waggoner at the Carnegie-Mellon University. This dye (sulfoindocyanine succinimidyl ester, CY5.18) covalently couples to lys residues specifically and is similar to carbocyanine dyes described previously (Mujumdar et al., 1989, 1992). Labeling of the Oat-25 Fab fragments was performed as follows: CY5.18 dye was dissolved in dry DMF and then immediately added to the Oat-25 fragments at a 2:l dye:Fab ratio and the mixture incubated for 30 min. Free unreacted dye chromophore was removed by passing the Fab and dye mixture over a 4 x 0.5 cm G-25 Sephadex column. Possible remaining unbound CY5.18 chromophore was further removed using a Centricon-10 by performing three separate concentration and dilutions steps. The approximate molar ratio of the number of CY5.18 dye molecules bound per Fab fragment was calculated from the absorption spectrum of the dye-labeled Fab fragE = ment based on the molar absorptivity of Fab‘ ( 6.8 x lo4; Der-Balain et al., 1988) and the carbocyanine dye CYS.18 (eos2nm = 2.0 x los; Dr. Mujumdar, personal communication). The calculated dyelprotein molar ratio for all measurements presented here was 0.8 dye molecule per Fab fragment. It is possible that Fab fragments had multiple-dye labels, and/or were labeled at different sites of the Fab. However, the conditions in the labeling procedure were adopted to ensure that the majority of Fab fragments contain only one dye IabellFab. Fluorescence measurements: instrumentation, Steadystate tluorescence measurements were recorded using a Shimadzu RF540 spectrofluorophotometer with a 620 nm long pass filter and the data transferred to an 80286 based IBM compatible computer using Lotus Measure. Fluorescence lifetimes were measured by the time-correlated single photon counting method (O’Connor and Phillips, 1984) using an Edinburgh Instruments 299T lifetime system with the addition of a pulsed-diode laser (Farrens and Song, 1991). The pulsed diode-laser (Hamamatsu Photonics PLP-01, 660 nm) was operated at between 1-3 MHz. The sample fluorescence was detected at right angles to the excitation laser by a red-sensitive PMT (Phillips XP2254B), followed by time-to-amplitude conversion (TAC; Ortec 567) and pulse shaping by constant fraction discriminator (Edinburgh CF 4000). The count rates were kept below 0.1% (1000 countls) to avoid pulse pile up. Light intensity of the diode-laser was varied using a linear wedge neutral density filter (Ealing Electro Optics). The TAC start-signal was generated by using the negative pulse (ca 10 ns) from the trigger-out on the back of the PLP-01 after processing it through the constant fraction discriminator to provide a better timing signal. Data were collected using a multi-channel pulse height analyzer (Ortec 5600) and the data then transferred to an 80286-based personal computer (WYSE). All lifetime decays were analyzed by computer software based on a non-linear least-squares reconvolution analysis method (Birch and Imhof, 1985). The software was purchased from Edinburgh Instruments. Fluorescent lifetime measurements: protocol. The Pr forms of phytochrome were measured while stirring the samples and with constant irradiation by 730 nm light to ensure that no Pfr accumulated by the measuring pulsed

~

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Photochrome phototransformation diode-laser. Lifetimes for the Pfr forms were measured from Fab-dye first bound to Pr, then converted to Pfr with 660 nm irradiation and lifetime immediately measured. Pulsed-diode laser had 660 nm output with a pulse-rate of 800 kHz. Emission was monitored at a right-angle through a monochromator set at 675 nm, and a linear polarizer set at 54.7" orientation to the vertically polarized excitation beam. All measurements took less than 15 min, ensuring that differences in Fab binding affinity between Pr and Pfr were minimized. The latter point was confirmed by monitoring the increase in steady-state fluorescence and fluorescence lifetimes after converting the Fab-dye boundPr solution to Pfr. No increase was observed at times less than 30 min. Forster energy transfer: theory and calculations. Energy from light absorbed by a fluorescent molecule can be transferred to another molecule through space by a nonradiative process (Forster, 1948). This process is the result of a direct coupling of the transition dipole of the fluorescent donor molecule to that of the accepting molecule, and can be described as (Fairclough and Cantor, 1978)

where T~~ is the lifetime of the donor-acceptor complex, and T~ is the lifetime of the energy donor with no acceptor present. Here the donor is the dye-labeled Fab and the acceptor is the phytochrome chromophore. Thus, T,., is the lifetime of the fluorescently labeled Fab bound to phytochrome, and 7, is the lifetime of the free, unbound dye-labeled Fab fragment. The efficiency (E) of energy transfer is related to the distance (R) between the energy donor chromophore and an acceptor chromophore by

Rearranging this equation, distances (R) between chromophores of interest in biological macromolecules is described by

(3) By definition, R, is the distance at which the efficiency of energy transfer (E) is 50%. R, is also dependent on a number of spectral factors unique to each set of donor and acceptor systems used, and is described by the following equation

~g = (8.79 x 10-5) * K2 * 1 - 4 * 4, * J,, (4) where q is the refractive index of the medium between the chromophores, 4, is the quantum yield of fluorescence for the donor (D), and K~ is an orientation factor between the transition dipoles of the donor and acceptor chromophores D and A (in most cases involving protein systems, K~ can be approximated by 2/3; Fairclough and Cantor, 1978). and J is the overlap integral (described below). To determine the R,, value, the spectral overlap of the absorption and fluorescence emission of the acceptor and donor must be determined. This value, called the J value, is described as

727

a computer spreadsheet program (Lotus 123) in 2 nm intervals. For Pr at 666 nm, E = 1.32 x lo5 was used. This value was scaled accordingly for all other wavelengths. For Pfr, a 87% conversion of Pr to Pfr was assumed, and the 4 values scaled accordingly. The overlap integrals obtained were J cm3 M-' for Pfr and 2.16 x = 7.12 X cm3 M-' for Pr. Ro values were then calculated using Eq. (4), q = 1.456, K* = 213, +D = 0.26 and the J values given above. The results are given in Table 1. In summary, to experimentally measure distances between two chromophores using the Forster energy transfer method, it is necessary to: (1) Calculate the Ro values for the pair of chromophores being studied, and (2) Measure the efficiency of energy transfer between the two donor and acceptor molecules.

RESULTS

The CY5.18 dye1Fab emission with the Pr and Pfr absorption spectra shown in Fig. 1 were used to calculate the overlap integral J . The long wavelengths (A4) and the excellent spectral overlap of the CY5.18 emission and the phytochrome absorption bands results in very high J values. Thus, the Ro distances calculated are also quite high. The Ro values for the CY5.18 labeled Oat-25 Fab fragment and phytochrome are 53 A for Pfr and 65 A for Pr (Table 2). To properly measure Forster energy transfer, all fluorescent donor molecules must have acceptors (Fairclough and Cantor, 1978). To determine what ratio of Fab-dye to phytochrome ensures that all dye-labeled Fab are bound to phytochrome, a titration of phytochrome with the Fab-dye fragments was performed (Fig. 2). At phytochrome1Fab-dye molar ratios greater than 5:1, no change in the steady state fluorescence quenching of the CY5.18 dye was observed. All measurements reported here used phytochrome1Fab-dye ratios of 6:l or 7:l to ensure that changes in fluorescence could be ascribed only to bound Fab-donors. The phytochrome chromophore was not seriously perturbed by the binding of the Fab-dye to the Nterminus as shown by the absorption spectrum in Table 1. Fluorescence lifetimes of free and phytochrome-conjugated Fab-CY5.18 dye Free

The larger the overlap of the fluorescent donor emission band (F(X)) with the absorption band (€(A)) of the acceptor molecule, the higher is the calculated overlap integral (J).J is also strongly dependent on the wavelength of light used, as can be seen by the X4 dependency.

J values were calculated by importing the fluorescence and absorption spectra shown in Fig. 1 into

Exp. number

Fab-dye (ns)

x2

Pr-Fab-dye Pfr-Fab-dye (ns) x2 (ns) x2

1.97 ? 0.01 1.3 1.56 ? 0.01 1.3 1.65 2 0.01 1.3 1.94 ? 0.02 1.3 1.55 ? 0.02 1.2 1.62 5 0.02 1.6 2.03 ? 0.01 1.6 1.57 2 0.01 1.2 1.65 2 0.02 1.1 Average 1.98 ns 1.56 ns 1.64 ns

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DAVIDL. FARRENS er al.

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650 750 Nanometer Figure 1. Normalized overlap spectrum of the CY5.18 carbocyanine dye fluorescence emission spectrum (covalently attached to the Oat-25 Fab fragment) with the absorption spectra of the Pr and Pfr forms of phytochrome, used to calculate the J value of Fab-dye/phytochrome donoracceptor pairs. 0.5 p M CY5.18 dye-labeled Oat-25 Fab fragment was used to record the fluorescence emission spectrum (excitation wavelength at 620 nm). The Pfr spectrum was corrected for -13% residual Pr (Kelly and Lagarias, 1985). All spectra were measured at 279-281 K, in 20 mM potassium phosphate buffer pH 7.8 containing 1 mM EDTA and 1 pg/mL leupeptin.

Fig. 3. A 1:l phytochrome1Fab-dye ratio was used to ensure that the phytochrome chromophore spectra were primarily from Fab-bound phytochrome. The 6:l phytochrome1Fab-dye ratio used in the energy transfer measurements ensures that all Fab is bound to phytochrome; however, at such a high phytochrome ratio, the free unbound phytochrome would dominate the absorption spectrum. The,,,E of the Pfr chromophore is a sensitive assay of perturbations to the phytochrome chromophore. Disruption of chromophore-protein interactions induces dramatic blue shifting of the Pfr absorption spectrum (Vierstra and Quail, 1983). The A,,, of Pfr

Phytochrome/Fab-dye ratio (moles/moles) Figure 2. Titration of CY5.18 dye-labeled Fab fragments with phytochrome. Steady state emission intensities were corrected for dilution of sample by each addition of phytochrome. Initial concentration of Fab-dye sample was 0.125 pM.Spectra were recorded using excitation at 640 nm and emission at 655 nm with a 620 nm long-pass filter. Other conditions were same as for Fig. 1.

600 Nanometer

400

800

Figure 3. Absorption spectrum of CY5.18-labeled Oat-25 Fab bound to the Pfr form of phytochrome. The Amax of Pfr is only slightly shifted, suggesting that the chromophore (and protein) has not been drastically perturbed by the binding of the Fab to the phytochrome. Other conditions were the same as for Fig. 1.

when the Fab-dye was bound to it was only shifted by 2 nm from the normal absorption maximum of 124 kDa native phytochrome and may be only an apparent shift due to the tailing of the high absorbance at 652 nm. This indicates that the phytochrome chromophore was not seriously disturbed by the binding of the Fab-dye, and the chromophore location and conformation can be considered to be that existing in the native protein state. After the Ro values were determined, and all Fab-dye donors were known to have acceptors, the lifetimes of the phytochrome bound and unbound Fab-dye were measured. Figure 4 shows typical fluorescence decay curves. The data from the Fab-dye phytochrome lifetime measurements are given in Table 1 below. All lifetimes represent independent measurements using different phytochrome samples from different isolations. Table 1 presents the fluorescence lifetimes of the dye-labeled Fab fragments unbound in solution and bound to phytochrome. A more complete description of conditions used is presented in the Materials and Methods section. The lifetimes in Table 1 were used to calculate the efficiency of energy transfer (E values). Using these measured values with the calculated Ro values and under the usual approximation of random orientation of the transition dipoles involved (Stryer and Haugland, 1967; Fairclough and Cantor, 1978), the apparent distance R between the N-terminus bound Fab-dye and the Table 2. Energy transfer efficiency, critical distance, and the distance (R)between the chromophore and the antibody binding site on the N-terminal chain

Pr-Fab-dye Pfr-Fab-dye Change in distance: (Rpr-RPfr)

Efficiency (E)

R,,

2 10% 17"/o

65 8,

53 12

A A

R 81 69 12

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Figure 4. (A) Fluorescence decay profile of CY5.18-labeled Oat-25 Fab fragment ( T ~= 466 ps, Ampl = 8.1%; i2 = 1.96 ns, Ampl = 91.9%). (B) Fluorescence decay profile of CY5.18-labeled Oat-25 Fab bound to Pr-phytochrome ( 7 , = 282 ps, Amp1 = 31.0%; T~ = 1.56 ns, Amp1 = 69.0%). (C) Fluorescence decay profile of CY5.18-labeled Fab bound to Pfr-phytochrome ( T ~= 418 ps, Ampl = 28.9%; = 1.65 ns, Ampl = 71.1%). Residuals of the profiles indicate a good fit of the decay curves. Solution conditions were the same as for Fig. 1. (Residuals are plotted in the lower panels as standard deviations; XSQ = chi square).

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DAVID L. FARRENS et al.

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phytochrome chromophore was determined. These results are given in Table 2. The fact that R changes by the same amount as Ro is merely coincidental, because these two distances were determined dominantly by lifetime measurements (Table 2) and by calculations (J and K), respectively, and the difference in energy transfer efficiency (Table 1) was always reproducible within the experimental errors indicated in Table 1. DISCUSSION

First, it should be noted that fluorescence contribution from phytochrome to the lifetime decays used to calculate energy transfer efficiencies is minimal or absent because the fluorescence quantum yield of phytochrome (Pr; Holzwarth et al., 1984) is about 100 times lower than that of the cyanine dye used; there is no detectable fluorescence from the Pfr form (Song et a f . , 1973). Energy transfer from the Fab-dye to the phytochrome chromophore is indicated by the decrease in fluorescence lifetimes observed upon binding of the Fab-dye fragment to phytochrome (Table 1). Similar results were obtained using steady-state fluorescence methods. There was 40-50% quenching of total steady state fluorescence of the Fab-dye upon binding to phytochrome, but the dynamic quenching as measured by fluorescence lifetime was substantially less (ca 20%) (data not shown). However, the difference between the Fab-Pr and Fab-Pfr steady state fluorescence intensities was approximately the same difference measured by the fluorescence lifetimes (21 and 17%, respectively). Thus the difference between the steady state and dynamic quenching is most likely attributable to energy transfer from the donor dye moiety to the phytochrome chromophore (vide infra). The CY5.18 carbocyanine dye used in these studies has a high fluorescence quantum yield (0.26; Mujumdar et al., 1992, personal communication), and a significant spectral overlap with the Pr form of phytochrome (Fig. 1). Unfortunately, currently no such far-red fluorescence emitting dyes for labeling proteins are available. To our knowledge, the R(, values for the Fab-Cy5.18 phytochrome pairs represent some of the largest values for a protein-dye label system yet reported. The CY5.18 carbocyanine dye suffers from one major drawback: the fluorescence lifetime of the dye conjugate is very low, measured with our system at 1.98 ns. Interpretation based on changes in the fluorescent lifetime requires that the instrument used be able to reliably and accurately measure very short lifetimes. Our single-photon counting system (Farrens and Song, 1991) has an instrument response function (IRF) of approx. 250 ps. Lifetimes of 1/4to 1/2of the IRF can be readily determined from lifetime data (O’Connor and Phillips, 1984). The changes in

lifetimes measured for the Fab-dye phytochrome complexes reported in Table 1 are much greater than this. Thus the changes of ca 500 ps reported here for the dye-labeled Fab fragments bound in the Pr and Pfr forms of phytochrome represent reproducible and significant differences. Two interpretations for the data presented in Table 1 are offered:

( I ) The distance between the Pfr chromophore and the Fab-dye bound to the N-terminus decreases A decrease in the distance between the phytochrome chromophore and the Fab-dye fragment bound to the N-terminus would increase the efficiency of energy transfer ( E ) between the Fabdye and the Pfr chromophore. This would compensate for the lower Ro distance for the Pfr Fab-dye complex. The conversion of Pr to Pfr results in a decrease of overlap for the CY5.18 emission with the phytochrome chromophore absorption (Fig. 1). Thus the overlap integral ( J ) decreases and the Ro distance of 65 A for Pr decreases to 53 A for Pfr. The efficiency of energy transfer ( E ) for the Fab-dye bound to Pfr should only have been 7% (lifetime = 1.84 ns), assuming no change in the distance ( R ) between the Fab-dye and the Pfr chromophore had occurred. This was not observed. (2) The binding of the dye-labeled Fab to phytochrome alters the environment around the fluorescent dye molecule (i.e. trivial fluorescence quenching) Trivial quenching must always be considered when doing fluorescence energy transfer measurements. For example, it is possible that change in lifetimes reflects reorientation of the chromophore in the Pr -+ Pfr phototransformation, as randomization of the chromophore transition dipoles may not be complete during the lifetime of the donor fluorescence. However, as stated above, the efficiency of energy transfer should be much less in the Pfr form. The uncertainty in distance introduced by the orientation factor K in a freely rotating-rigid acceptor system is relatively small, usually less than 20% (Stryer, 1978). To attempt to rule out possibility (2),fluorescence anisotropy studies were performed on the free Fabdye and the Fab-dye bound to phytochrome. However, it was not possible to obtain the anisotropy of the dye when the Fab-dye was bound to phytochrome. This is because during the long times necessary for the anisotropy measurements (ca 2 h) it was impossible to exclude a small amount of the 730 nm back-converting light from impinging on the measuring PMT. Thus an unacceptable amount of background noise was present in the Fab-dye phytochrome anisotropy results. The difficulty of measur-

Photochrome phototransformation

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ing anisotropy of a molecule with very short life- ing suggests that the N-terminus-chromophore times (in this case less than 2 ns) and the nature of interaction has not been disrupted (Fig. 3). A rapid the experimental system used here made it imposs- dark reversion of Pfr to Pr caused by the binding ible to obtain anisotropy data with good statistical of full size Oat-25 was not observable with the precision. Oat-25 Fab fragments within the time period of We feel that the results in Table 1 are more measurement required. If it had occurred to any consistent with interpretation ( l ) , and that the Pfr extent, movement of the chromophore would be chromophore moves toward the N-terminus bound underestimated accordingly as a consequence of Pfr Fab-dye fragment upon Pr -+ Pfr photo- potentially reverting during the time period of transformation. However, explanation (2) cannot be measurements. Also, chromophore movement is completely ruled out. Further support for expla- not affected by the N-terminal chain, as the chromonation (1) is provided by the following observation: phore reorients with or without the presence of the two lifetimes are observed for all of the fluorescently N-terminus (Sundqvist and Bjorn, 1983; Ekelund et labeled Fab fragments; a short component al., 1985). This indicates that a possible effect of (300-600 ps) corresponding to roughly 15-30% of Fab on a a-helical folding of the N-terminus does the total fluorescence, and a longer component not affect chromophore movement. Finally, it should be noted that the relative move(1.55-2.0 ns) which corresponds to 70-85% of the total fluorescence. The origin of the shorter lifetime ment of the chromophore referred to here may component is unknown. It may be due to two differ- include a small contribution from the reorientation ent sites of labeling on the Oat-25 Fab antibody of the chromophore Q,- transition dipole (Pr vs fragment, two distinct conformers of the Pfr), contrary to the assumption (K’ = 2/3) used to antibody-dye conjugate, or two inherently different calculate the Ro values (vide supra). lifetimes of the CY5.18 dye when bound to a protein (Fab). The inter-subunit energy transfer from the chromophore to the N-terminal chain within the CONCLUSION dimeric phytochrome molecule is negligible, if any, Forster energy transfer from fluorescently labeled because the distance between the chromophore of one monomer subunit to the N-terminus of the anti-phytochrome Fab fragments was used to deterother monomer subunit is too far (Jones and Erick- mine changes in distance between the chromophore son, 1989; Tokutomi et al., 1989) for any significant and the N-terminus in the Pr -+ Pfr transformation of phytochrome. The results strongly suggest that energy transfer. The amplitude of this short lifetime component the phytochrome chromophore moves relative to ( O h amount) changed little upon binding to phyto- the N-terminus region (where the dye-labeled Fab chrome but the lifetime behaved very similarly to is bound) in the Pr to Pfr phototransformation. Previous work by a number of laboratories has that of the longer lived component. Using the short lifetime component to calculate the R distances indicated that the Pfr chromophore interacts with yielded values of R = 66 A for Pr, and R = 56 8, for the N-terminus in phytochrome (Vierstra and Quail, Pfr. The difference between these two R distances 1983; Chai et al., 1987). It has also been shown that (10 A) upon Pr to Pfr transformation is similar to the chromophore in phytochrome reorients upon Pr the decrease in R (12 A) observed using the long to Pfr photoconversion (Sarkar and Song, 1982; lifetime decay component. Thus calculation of the Ekelund et al., 1985; Tokutomi and Mimuro, 1989). change in R distance using the short lifetime decay The present work is consistent with these previous components also supports the conclusion that the findings. We conclude that upon Pr + Pfr photodistance decreases between the chromophore and transformation, a significant movement of the the N-terminus upon Pr to Pfr phototransformation. chromophore occurs which decreases the distance The movement of the N-terminal chain toward the between the phytochrome chromophore and the NPfr chromophore is also possible, but we feel that terminus by approximately 10-12 A. The use of fluorescently labeled Fab fragments to the relative movement of the chromophore toward map out changes in protein distance by fluorescence the N-terminus in Pfr is more likely because the chromophore reorientation induced by the photo- energy transfer studies has not been previously transformation is independent of the presence of reported. Because of the large number of Fab with the N-terminal chain (Sundqvist and Bjorn, 1983; known binding sites, with further refinement this technique should see wider use in future biological studies. Ekelund et al., 1985). Full size Oat-25 destabilizes Pfr if the antibody is bound first to Pr, as ,,A shifts from 730 to 720 nm Acknowledgemenrs-This work was supported by a grant (Cordonnier et al., 1985). However, the same was from USPHS NIH (GM36956). The subnanosecond lifenot happening in a significant way when only the time equipment used for the present study was purchased with grants from NSF DIR 8907375 and the Center for Fab fragment was used and the measurements were Biotechnology, University of Nebraska. We thank Promade within 15 min. The small shift in A,,, fesor A. Waggoner and Dr. R . B. Majumdar for a gen(728 nm) for the Pfr species upon Oat-25 Fab bind- erous gift of the cyanine dye used in this study.

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