(Received for publication, October 6, 1994; and in revised form, November 29, 1994)
From the
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.
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
( 380 nm) and for free all-trans-retinal
(
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.
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) .
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) .
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.
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.
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).
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.
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) .
This is paper 11 in the series ``Structure and Function in Rhodopsin.'' The preceding paper in this series is (1) .