©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Function in Rhodopsin
MEASUREMENT OF THE RATE OF METARHODOPSIN II DECAY BY FLUORESCENCE SPECTROSCOPY (*)

(Received for publication, October 6, 1994; and in revised form, November 29, 1994)

David L. Farrens (§) H. Gobind Khorana (¶)

From the Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An increase in fluorescence is observed upon light activation of bovine rhodopsin. The rate of increase is monoexponential (t = 15.5 min) at 20 °C in 0.1% lauryl maltoside, pH 6.0, and parallels the rate of decay of metarhodopsin II. We show that the increase in fluorescence is due to the release of free retinal, which no longer quenches the tryptophan fluorescence. An extrinsic fluorescence reporter group, pyrene maleimide, attached to bovine rhodopsin also shows an increase in pyrene fluorescence on illumination. The rate of increase in pyrene fluorescence matches the rate of increase in tryptophan fluorescence. This result has been used to develop a micromethod, requiring on the order of 1 µg of rhodopsin, for measurement of metarhodopsin II decay. An Arrhenius plot derived from the fluorescence assay shows the energy of activation barrier for retinal release from rhodopsin to be 20.2 kcal/mol in 0.1% dodecyl maltoside at pH 6.0.


INTRODUCTION

Rhodopsin, the mammalian photoreceptor, undergoes, on illumination, isomerization of the retinal from the 11-cis to the all-trans form. This event causes the formation of a series of transient intermediates in the protein(2) . One of these, metarhodopsin II, initiates a cascade of biochemical reactions which leads to the closing of the cation conductance channels in the plasma membrane of the rod cell(2, 3) . The rate of metarhodopsin II decay is usually determined by measuring the decrease of the Schiff base linkage between retinal and the protein after acidification (4, 5) and not directly by absorption spectroscopy since the absorption maxima for metarhodopsin II ((max) 380 nm) and for free all-trans-retinal ((max) 387 nm) are similar. We show here that following illumination of rhodopsin there occurs an increase in tryptophan fluorescence (Fig. 1) and that the rate of this increase parallels the rate of decay of metarhodopsin II. That the increase in fluorescence is due to the release of free retinal has been confirmed by the following experiments. 1) Rhodopsin carrying the fluorescence reporter group, pyrene maleimide, at cysteine 140, on illumination shows an increase in pyrene fluorescence at a rate that matches the rate of increase of tryptophan fluorescence on metarhodopsin II decay. 2) The increase in tryptophan fluorescence is abolished when exogenously added 11-cis-retinal is present during metarhodopsin II decay. 3) The presence of hydroxylamine, which cleaves the Schiff base linkage in metarhodopsin II, during illumination of rhodopsin accelerates the increase in tryptophan fluorescence. The fluorescence increase provides a convenient method for measuring the rate of metarhodopsin II decay. The method is simple, sensitive, and requires only around 1 µg of rhodopsin.


Figure 1: Secondary structure model of rhodopsin showing the three domains: cytoplasmic, membrane-embedded, and intradiscal. The boundaries between the domains shown by the horizontal lines are approximate. Single-letter abbreviations are used for the amino acids. The 5 tryptophans responsible for the fluorescence are highlighted by circles around the letters. The tryptophans, presumably, are all in the membrane-embedded domain. The site of the protonated retinyl Schiff base (Lys-296, boxed with plus sign outside) and the Glu-113, the counterion (boxed with a minus sign outside) are shown.




EXPERIMENTAL PROCEDURES

Materials

Bovine retinas were from J. A. Lawson Co. (Lincoln, NE). 11-cis-Retinal was a generous gift of Professor R. Crouch (Medical University of South Carolina and the National Eye Institute). Pyrene maleimide was purchased from Molecular Probes (Eugene, OR). Dodecyl maltoside was purchased from Anatrace (Maumee, OH).

Methods

Purification of ROS^1Rhodopsin

ROS membranes from frozen bovine retinas were prepared under dim red light according to Wilden and Kuhn(6) . ROS rhodopsin was solubilized (1% dodecyl maltoside) and purified on ConA-Sepharose (Pharmacia Biotech Inc.) by chromatography(1, 7) .

Rate of Metarhodopsin II Decay Measured by Surviving Retinyl Schiff Base

The rate of retinal release on metarhodopsin II decay was determined by following the decrease in protonated retinyl Schiff base as measured at 440 nm after acidification(4, 5) .

Expression and Purification of Rhodopsin from the Cysteine Mutant

The cysteine mutant rhodopsin gene (C316S, C322S, C323S) containing Cys-140, the only reactive cysteine, has been described previously(8, 9) . This was used in the present work and is designated the cysteine mutant. It was expressed in COS cells, and the protein was purified on anti-rhodopsin 1D4-Sepharose(10, 11) .

Pyrene Maleimide Labeling of the Cysteine Mutant Rhodopsin

A stock solution (23 mM) of PM was freshly prepared in dimethyl sulfoxide. A 1-µl aliquot of this solution was added to a suspension of 2 ml of 1D4-Sepharose containing 40 µg of bound cysteine mutant rhodopsin in 10 ml of buffer A (0.1% dodecyl maltoside, in 10 mM MES, pH. 6.0). The mixture was nutated overnight in the dark at room temperature. The Sepharose beads were collected by centrifugation and washed five times (10 ml each time) with buffer A. The PM-derivatized rhodopsin mutant, now designated PM-rhodopsin, was eluted with the carboxyl-terminal octadecapeptide (300 µl of 40 µM solution in buffer A). The extent of labeling was 0.6 mol of PM/mol of rhodopsin as determined from the absorption spectrum using (max) 500 nm, 40,600 and (max) 340 nm, 36,000.

Steady-state Fluorescence Measurements

These were performed on a SPEX 212x Fluorolog Instrument (Spex Industries Inc., Edison, NJ). Data manipulations were performed using the SPEX software or with Sigma Plot (Galactic Industries). Rhodopsin samples (150-200 µl of 0.1-1 µM) were used in temperature-controlled microfluorescence cuvettes. Low excitation light intensities were used throughout to prevent photolysis of rhodopsin. Slit settings for the fluorometer were 0.2 nm for the excitation and 12 nm for the emission measurements. Rhodopsin samples were excited at 295 nm to probe tryptophan fluorescence selectively. For the time base measurements, data were recorded at 330 nm for 2 s at 30-s intervals, with the excitation shutter closed between acquisitions to minimize exposure to the measuring beam. No detectable photobleaching of rhodopsin sample was observed under these conditions. The PM-rhodopsin was excited with 340 nm light, and emission was monitored at 384 nm. For conversion to metarhodopsin II, the samples were bleached as described(1) . The half-lives () of the fluorescence increases were fit to a single exponent using the computer program SigmaPlot (Jandel Scientific). All fits showed an r^2 value >0.95.

Fluorescence Lifetime Measurements

Fluorescence lifetimes were measured at the University of Nebraska with a time-correlated single photon counting Edinburgh Instruments 299T lifetime system(12) . Excitation was with a hydrogen flash lamp operated at 40 kHz. For tryptophan fluorescence, the excitation wavelength was selected as 292 nm using a monochromator, and fluorescence emission was monitored at 347 nm using an interference filter. Opsin was formed by bleaching rhodopsin with white light for 1 min and then waiting 2 h before measuring.

NaBH(4)Reduction of Rhodopsin

The fluorescent N-retinylidene opsin species was produced by reduction of the Schiff base in rhodopsin with NaBH(4) using the procedure of Bownds and Wald(13) , modified as follows. An ROS rhodopsin solution (400 µl of 0.5 µM rhodopsin in buffer A) containing 100 µl of 1 M KPO(4), pH 7.0, was photobleached and then treated with 1 mg of NaBH(4). The absorption spectrum showed that the reduction to the N-retinylidene opsin ((max), 330 nm) was complete within a few min.


RESULTS

Quenching of Tryptophan Fluorescence by Retinal

Tryptophans in dark state rhodopsin exhibit only weak fluorescence, with an emission maximum centered at approximately 330 nm (Fig. 2A). Following illumination, there is a a small decrease in the fluorescence emission intensity (Fig. 2A), presumably because of a more efficient energy transfer from tryptophans to retinal in the metarhodopsin II state. Fluorescence then begins to increase; 2 h later when metarhodopsin II decay is complete, the tryptophan fluorescence is more than 4-fold over that observed in dark state rhodopsin (Fig. 2B). Similarly, Table 1shows that the fluorescence lifetimes of tryptophans increase upon photoconversion of rhodopsin to opsin.


Figure 2: Panel A, fluorescence emission from rhodopsin in the dark and immediately after photobleaching (metarhodopsin II). Panel B, fluorescence emission from rhodopsin in the dark and from opsin formed on decay (2 h) of metarhodopsin II. The measurements were at 20 °C in buffer A. Excitation was at 295 nm.





To confirm that the quenching of tryptophan emission in rhodopsin in the dark state is caused by energy transfer to retinal, we reduced the retinyl Schiff base linkage in rhodopsin to the fluorescent derivative. The excitation spectrum of the N-retinylidene opsin species monitored at 490 nm showed an excitation band at 330 nm and an extra excitation band at 280 due to energy transfer from the aromatic groups to the retinylidene group (not shown). This result is in agreement with the previous report by Ebrey(14) .

Increase in Fluorescence as a Function of Metarhodopsin II Decay

The rate of increase in tryptophan fluorescence from photobleached rhodopsin is compared in Fig. 3with the rate of decay of metarhodopsin II as measured by the decrease in the protonated Schiff base concentration. The two processes occur in parallel, with the of fluorescence increase at 15.5 min, whereas that of Schiff base hydrolysis at 15.9 min. Thus, the fluorescence increase occurs as a consequence of free retinal formation.


Figure 3: Kinetics of fluorescence increase at 330 nm and of Schiff base hydrolysis after photobleaching of bovine rhodopsin. The values observed for the two processes were 15.5 and 15.9 min, respectively. Both samples were in buffer A at 20 °C, and fluorescence excitation was at 295 nm. The amount of the protonated Schiff base surviving at different times was measured by absorption at 440 nm as described previously(4, 5) .



The Presence of 11-cis-Retinal during Metarhodopsin II Decay Abolishes Fluorescence Increase

Fig. 4shows the effect of exogenously added 11-cis-retinal on fluorescence increase following photobleaching. Curve 1 is the control showing normal fluorescence increase as a function of time on metarhodopsin II decay. Curve 2 shows that the addition of 11-cis-retinal completely arrests the increase in fluorescence. As has been shown recently(1) , when 11-cis-retinal is present during metarhodopsin II decay, the opsin formed binds 11-cis-retinal to regenerate the native rhodopsin. The formation of the latter ensures the quenching of tryptophan fluorescence. As a consequence no increase in tryptophan fluorescence is seen in the experiment of curve 2 of Fig. 4.


Figure 4: Effect of exogenously added 11-cis-retinal on fluorescence increase following rhodopsin illumination. Curve 1, kinetics of fluorescence increase under standard conditions; curve 2, effect of the addition of a 3-fold molar excess of 11-cis-retinal at the time shown. The samples contained 1 µM rhodopsin in buffer A at 20 °C. Excitation was at 295 nm, and emission was measured at 330 nm.



Effect of Hydroxylamine on Fluorescence Increase

The increase in fluorescence is greatly accelerated when rhodopsin is bleached in the presence of 10 mM hydroxylamine (Fig. 5). Although rhodopsin in the dark is stable to hydroxylamine at neutral pH, the retinyl Schiff base in metarhodopsin II is accessible to hydroxylamine and is rapidly cleaved by it(15) . This results in the rapid increase in fluorescence as seen in Fig. 5, in the presence of hydroxylamine.


Figure 5: Effect of the presence of hydroxylamine during photobleaching of rhodopsin on fluorescence increase. Curve 1, fluorescence increase from photobleached rhodopsin under standard conditions; curve 2, Fluorescence as measured from rhodopsin sample photobleached in the presence of 10 mM hydroxylamine. Both samples were at 20 °C in buffer A. Excitation was at 295 nm, and emission was measured at 330 nm.



Pyrene Fluorescence Increase on Bleaching of PM-Rhodopsin

PM attached to rhodopsin has been reported to exhibit fluorescence energy transfer to retinal(16) . Upon photobleaching of PM-rhodopsin, the pyrene fluorescence initially decreases (not shown), as for rhodopsin itself (Fig. 1). The fluorescence then increases as shown in Fig. 6. Fig. 6A shows the fluorescence increase from the tryptophan residues (295 nm excitation, 330 nm emission); Fig. 6B shows the fluorescence increase from the pyrene group (340 nm excitation, 384 nm emission). In both cases, fluorescence increases at virtually identical rates. Thus, the rate of metarhodopsin II decay as measured by the pyrene fluorescence increase is the same as measured by the increase of intrinsic tryptophan fluorescence.


Figure 6: Comparison of the rates of fluorescence increase from photobleached rhodopsin (panel A) and PM-rhodopsin (panel B). In panel A, excitation was at 295 nm, and emission was measured as above at 330 nm. Panel B, increase in pyrene fluorescence from PM-rhodopsin. Excitation was at 340 nm; emission was measured at 384 nm. Both samples contained 0.1 µM PM-rhodopsin in buffer A at 20 °C. The values for fluorescence increase are indicated and were determined by a single exponential fit (solid line) of the data (dots).



Activation Energy for Metarhodopsin II Decay Measured by Fluorescence Increase

The rate of fluorescence increase from photobleached ROS rhodopsin in buffer A was measured at nine different temperatures, ranging from 3 to 36 °C. The rate of fluorescence increase was temperature-dependent (Fig. 7). The linear Arrhenius plot of the rates yielded an energy of activation for metarhodopsin II decay of 20.2 kcal/mol.


Figure 7: Arrhenius plot of the rates of fluorescence increase from photobleached rhodopsin at different temperatures. All samples were in buffer A, the temperature ranging from 3 to 36 °C. Excitation was at 295 nm, and emission was measured at 330 nm. Data were fit to a straight line using SigmaPlot.




DISCUSSION

We first confirmed the earlier conclusions of Ebrey (14) and Kropf (17) that fluorescence energy transfer occurs from tryptophan residues in rhodopsin to the retinal chromophore in the dark state rhodopsin molecule. Presumably, all five tryptophans located in different helices in the membrane-embedded domain (Fig. 1) are able to transfer fluorescence energy efficiently. Further, we found only a small change in the tryptophan fluorescence emission upon formation of metarhodopsin II per se, and the subsequent increase in fluorescence also observed previously (21, 22) was a result of the release of free retinal. This increase occurred with a monoexponential rate that matched the rate of retinal release as determined by the surviving protonated Schiff base (Fig. 3).

The conclusion that the release of free retinal is the cause of fluorescence increase was supported by additional observations. First, when 11-cis-retinal was present during metarhodopsin II decay, the increase in fluorescence was abolished. This is consistent with the observation that following the release of all-trans-retinal on metarhodopsin II decay, correctly folded opsin is formed which binds 11-cis-retinal to form the native rhodopsin(1) . Second, hydroxylamine, which is known to cleave the retinyl Schiff base linkage in metarhodopsin II, accelerated the increase in fluorescence on rhodopsin illumination (Fig. 4).

To confirm that the increase in tryptophan fluorescence on photobleaching is due to the removal of the quencher, retinal, and not to some other change in the tryptophan environment, we attached to rhodopsin a pyrene fluorescence reporter group, also able to transfer energy to retinal. Upon photobleaching of the PM-rhodopsin, the pyrene fluorescence increased at a rate identical to the rate of tryptophan fluorescence increase from the same derivative (Fig. 6).

The thermodynamics of the fluorescence increase were investigated by photobleaching rhodopsin samples at nine different temperatures (3-36 °C) and measuring the rates of their fluorescence increase. The Arrhenius plot of the results (Fig. 7) indicates an activation energy of 20.2 kcal/mol. This value for metarhodopsin II decay is very similar to the activation energy of 20 kcal/mol determined previously for bovine rhodopsin in solution (18) and that of 16% 5 kcal/mol determined for rat rhodopsin in situ(19) .

We anticipate a number of applications of the present method for measuring metarhodopsin II decay in the study of a variety of rhodopsin mutants currently under investigation in this laboratory. For example, some rhodopsin mutants are deficient in activating the G-protein transducin. The assay described here provides a rapid and convenient way to determine if these mutants are deficient due to an altered rate of metarhodopsin II decay. Further, tryptophan fluorescence measurement with and without exogenous retinal addition can be used as an alternate way to determine if retinal has been inserted into the retinal binding pocket. This is of particular advantage where retinal binding is accompanied by only slight opsin shifts(20) .


FOOTNOTES

*
This work was supported in part by Grant GM28289 from the National Institutes of Health. The preceding paper in this series is (1) . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This is paper 11 in the series ``Structure and Function in Rhodopsin.'' The preceding paper in this series is (1) .

§
Recipient of National Eye Institute Postdoctoral Fellowship 1-F32-EY06465.

To whom correspondence should be addressed: Dept. of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-1871; Fax: 617-253-0533.

(^1)
The abbreviations used are: ROS, rod outer segment rhodopsin; G-protein, guanine nucleotide-binding regulatory protein; PM, pyrene maleimide; MES, 4-morpholineethanesulfonic acid.


ACKNOWLEDGEMENTS

We have greatly benefited from discussions with Professor U. L. RajBhandary (M. I. T.), Professor Mark Krebs (University of Wisconsin), and with our colleagues in this laboratory. We also thank Professor Pill-Soon Song, University of Nebraska, for discussions and for allowing us to measure the fluorescence lifetimes in his laboratory. The fluorescence spectra were measured in the Massachusetts Institute of Technology Laser Biomedical Research Center (a National Science Foundation Regional Instrumentation Facility). The sustained and enthusiastic assistance of Judy Carlin in the processing of this manuscript is gratefully acknowledged.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.