(Received for publication, August 7, 1995; and in revised form, October 18, 1995)
From the
The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge.
Bacterioopsin (BO) ()tunes the absorption maximum
(
) of a retinal protonated Schiff base (PSB) from
440 nm in methanol to 568 nm in bacteriorhodopsin (BR). The mechanism
of wavelength regulation in BR and in retinylidene proteins in general
has been a subject of intensive investigation. Nakanishi et
al.(1) introduced the term opsin shift (OS) to refer to
the energy difference (in cm
) between the absorption
maximum of PSB in methanol and that in the protein. Three factors have
been suggested to contribute to the 5100 cm
OS of
BR. (i) The conversion of the twisted conformation of the
-ionone
ring/polyene chain of the retinal PSB in solution (2) to a
coplanarized conformation (3) induced by the protein
contributes 1200-1300 cm
(4) . This
mechanism has been widely accepted. (ii) A weakened PSB/counteranion
association, either by increasing interionic distance or the solvation
of counterions, may further shift the absorption maximum to longer
wavelengths(5, 6) . (iii) Negative charges, or in a
more general sense, the polarizable groups and permanent dipoles
distributed along the conjugation chain may stabilize the excited state
and cause a red shift(7) .
There have been diverse estimates
of the counterion contribution to the OS of BR. 11,12-Dihydro-BR
analog, the chromophore structure of which is very similar to the
native one, exhibits an OS of 1400 cm(8) .
Solid state
C and
N NMR studies suggested a
value of 2000 cm
from the contribution of the
counterion (9) . Albeck et al.(10) and Hu et al.(11) suggested that a weakened counterion and
the ring/chain coplanarity together would produce an OS value in model
compounds nearly comparable with that in BR. Site-directed mutagenesis
of BR (12, 13) and solid-state NMR studies (14) indicated that both Asp-85 and Asp-212 are involved in
forming a complex counterion environment. In model compound studies,
introduction of a second negative charge near the Schiff base produces
a blue shift of 600-950 cm
(15) . In a
mutant BR (D85N/D212N) lacking both protein counterions, halide
functioned as a surrogate counterion so that the protein complexed with
chloride exhibited the same OS (
5100 cm
) as in
native BR(12, 13) . Although model PSB was measured in
the polar solvent methanol, which may cause significant solvation of
the Schiff base counterion(15) , the association of an anion to
a protein cation may still be different from the model system. In
D85N/D212N BR, one can argue that the halide may be more or less
separated from the PSB compared with solution model compounds. One
possible situation is that the halide is well separated from the Schiff
base charge and causes an OS by a similar ``weakened
counterion'' mechanism as in the native BR. This assumption does
not contradict to the correlation between the size of halide
(Cl-, Br-, I-) and the amount of red shift in the
mutant BR (12, 13) if one assumes a water molecule is
bound between the PSB positive charge and the counterions. However,
this correlation also fits the assumption that the chloride ion is
closely associated with the PSB positive charge. A closely associated
counterion was predicted by Honig et al.(16) on the
basis of energetic considerations. In contrast to the inorganic
counteranion in model PSB studies(15) , the dynamic protein
structure, in principle, allows associated counterions to fluctuate,
thereby allowing them to contribute to the OS of BR.
For convenience of computations, the external point charge model used a point charge to represent either a negative charge in a salt bridge or a negative end of a protein dipole. Mutagenesis studies (17) and two photon spectroscopy studies (18) argue against a discrete charge in the binding site but not against the local electrostatic fields, which would fulfill the conditions of the original point charge model(1) . Model studies(19, 20) , genetic analysis(21) , mutagenesis(22, 23, 24) , and resonance Raman (25) studies on various rhodopsin pigments all suggest that wavelengths can be regulated by polar side chains along the polyene chain.
To further test for the effective chromophore/protein
interactions other than the widely accepted ring/chain coplanarization (3, 4) and counterion
mechanisms(9, 11, 14) , we synthesized two
retinal analogs (I and II shown in Fig. 1),
which have a saturated 13-14 bond so that the counterion is
isolated from the conjugation system and hence can no longer contribute
to the OS. Furthermore, analog II possesses a planar
conformation in solution so that -ionone ring/polyene chain
coplanarization will not contribute to the OS.
Figure 1: I, 13,14-dihydro-all-trans-retinal; II, 3,7,11-trimethyldodeca-4,6,8,10-tetraenal.
Analog I absorbs at 289 nm in ethanol with a diffuse
bell-shaped spectrum (Fig. 2A, dotted line)
due to C-6-C-7 distortion(28) . It binds to apomembranes (27) containing BO and forms BR(I) absorbing at 328 nm
with distinct vibrational fine structure (Fig. 2A, solid lines). The OS in this pigment is 4110
cm. The red shift and the development of fine
structure indicate that the chromophore is tightly bound and that the
analog is forced to adopt a ring/chain coplanarized
conformation(8) . The analog evidently binds to the same site
and assumes analogous retinal/protein interactions as in native BR
since it results in a 270-fold reduction in the rate of incorporation
of native retinal to this pigment (data not shown). To dissect the
coplanarity-induced shift from other possible contributions in
BR(I), analog II with the same chromophore length but
a planar conformation was studied. Analog II absorbs maximally
at 307 nm and exhibits distinct vibrational fine structure in methanol (Fig. 2B, dotted line). The red shift in
analog II compared with analog I and its structured
spectrum indicate a planar conformation. Although the lack of the
native
-ionone ring in II may affect the shift in the
resulting pigment when incorporated into BO, a red shift is expected if
a spectral tuning mechanism other than the counterion and the
ring/chain coplanarity exists. Analog II binds to BO and
generates BR(II) absorbing maximally at 321 nm (Fig. 2B, solid lines). The OS in this pigment
is 1420 cm
, and the fine structure prominent in the
free analog is considerably blurred. The occupancy of the native
binding site by the analog was confirmed by an 11-fold inhibition of
the native retinal incorporation.
Figure 2:
A 5-µl ethanolic solution of compound
I (3 10
M) (A) or compound
II (1
10
M) (B) was added
to a 3-ml apomembrane suspension containing BO at pH 7.0 and 23 °C.
Absorption spectra (solid lines) were recorded at 2, 14, 30,
and 45 min (A) and 2, 5, 9, 12, and 40 min (B) after
analog addition as seen from the rise at 328 (A) and 321 nm (B), respectively. Unreconstituted apomembranes to which was
added the same volume of ethanol were used as a reference. Spectra of
analogs in ethanol are shown as dotted
lines.
There are two ways to calculate the OS of BR(I) in
the absence of counterion and coplanarization contributions. (i)
Subtraction of the amount of red shift caused by coplanarity
( -
= 2030
cm
) from the OS of BR(I), 4110
cm
, indicates an additional OS of 2080
cm
in BR(I). (ii) The difference between
OSs of BR(I) and BR(II) is 2690
cm
. By either calculation there exists additional OS
resulting from mechanisms other than the counterion and ring/chain
coplanarization. The
600 cm
difference between
the two methods of calculation is not surprising. The lack of the
-ionone ring may alter the orientation of the analog II in the BR retinal binding site. In other words, a hypothetical
prelocked, ring/chain planarized 13,14-dihydroretinal analog may give a
higher OS than analog II upon binding to BO. The instability
of analog II in the binding site is indicated by two
experimental findings. (i) Analog I when bound to BO retards
the incorporation of the native retinal by 270-fold while analog II does so by only 11-fold. This effect may result from the lack of
the native
-ionone ring on the chromophore and the partially
impaired retinal ring/protein interactions in BR(II). (ii) The
distinct vibrational fine structure observed in BR(I) is
diffused in BR(II), also indicating analog II is more
flexible due to the lack of native interactions in the binding pocket.
Therefore, we consider 2080 cm
as a better estimate
of the OS in BR(I) in the absence of counterion and
coplanarization effects. The possibility of intramolecular
through-space interaction has been previously excluded experimentally
by comparing the absorption maxima of dihydro- and tetrahydroretinal
analogs and pigment analogs (29) . Therefore, this OS
demonstrates the existence of a red shift mechanism in the absence of
the counterion and the coplanarization contributions.
We find two
possibilities particularly attractive in explaining the source(s) of
the additional red shift. (i) An aromatic retinal binding pocket was
originally proposed by Braiman et al.(40) and
Rothschild et al.(41) based on Fourier transform
infrared spectroscopic studies. From the recent atomic resolution BR
structural model(30) , the retinal chromophore is tightly
sandwiched by an aromatic pocket including Trp-182 above, Trp-86 and
Trp-189 under, and Tyr-185 on the side of the retinal. In bulk
solutions, a polarization-induced shift on absorption results from
momentary polarization in a polarizable solvent induced by the
transition dipole of the solute(31) . Since photoexcitation
produces a large change in retinal dipole moment(32) ,
polarizable solvents red shift the of PSB by 2390
and 2030 cm
(19, 20) . The
preorganized aromatic retinal binding pocket in BR would produce more
effective stabilization for the Franck-Condon state of the chromophore
within its lifetime compared with effects observed in bulk solution. An
early study of the energy transfer between retinal and tryptophans
indicated the close interactions between them(42) . Polarizable
aromatic compounds have also been shown to effectively stabilize
positive charges(33, 34) . However, since tryptophans
are expected to play a key role in fixing the backbone of retinal in
addition to their role in wavelength determination, tryptophan mutants
of BR do not provide a clear test of this mechanism. (ii) Second, fixed
polar groups around the polyene chain of retinal may be located in such
a way that they can effectively stabilize the excited state or
destabilize the ground state of the chromophore. This mechanism is
theoretically sound(7, 35) , and experimental evidence
has begun to accumulate. For example, hydroxyl groups have been shown
to play a key role in wavelength regulation in human cone pigments by
several
groups(21, 22, 23, 24, 25) .
Incorporating either of the two mechanisms into the BR wavelength
regulation scheme can satisfactorily account for our results as well as
a number of previous observations of red shifted retinoid chromophores
in the absence of PSB formation. Large OS values (3000-4700
cm) have been observed in non-covalent complexes
between BO and retinal analogs with one less double bond (36) .
The BR reconstitution intermediate, which was shown to be
non-covalently bound to BO exhibits an OS of 3100
cm
(37) . The BR photocycle intermediate
M
contains a deprotonated Schiff base chromophore, yet
its OS is 3350 cm
. All-trans-retinal cannot
covalently bind to the K216C mutant BO protein in a PSB form, but the
OS of this non-covalent species is 3330
cm
(38) . All-trans-retinal can be
complexed non-covalently with the dried membrane of BO and a mutant BO
K216G and forms a common complex absorbing at 470 nm (OS 4360
cm
)(39) . All of these opsin shifts greatly
exceed the ring/chain coplanarization effect of 1200-1300
cm
deduced from the
difference
between native retinal and planar 1,1-demethyl retinal or the
(6-S)-trans-locked retinal.
Chromophore/protein interactions that enable the significant red shift of BR are too complex to be modeled by simple synthetic model compounds. A retinal analog approach is advantageous in that artificial retinals are used to explore the actual real protein binding site. However, we should keep in mind that retinal analogs may be affected differently than the native chromophore. For this reason, the OSs reported here for BR(I) and BR(II), although revealing an effective wavelength regulation mechanism in the retinal binding site, do not quantitatively measure the amount of OS in native BR from this mechanism. Nevertheless our studies demonstrate that some protein/chromophore interactions other than those involved in retinal ring/chain coplanarization and the weakened counterion make contributions to the BR red shift, and these interactions are located along the retinal polyene chain. These interactions are likely to be between retinal and the polar or polarizable protein side chains located in the retinal binding site. Our results suggest that the spectral tuning in BR is achieved by synergistic chromophore/protein interactions including ring/chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and the weakened counterion interaction near the Schiff base positive charge.