 |
INTRODUCTION |
Photoactive yellow protein
(PYP)1 is a photoreceptor
that has been found in several purple bacteria (1). The first, and so
far best studied example for this group of blue light receptors, was
found in Ectothiorhodospira halophila (E-PYP) (2). The chromophore, responsible for the photophysical properties of PYP, is
4-hydroxy-cinnamic acid that is bound to Cys-69 via a thiol-ester linkage (3, 4). The crystal structure of this small protein, consisting
of 125 amino acids, has been solved to 1.4-Å resolution (5) and shows
an
/
-fold, which has become the prototype for the folding of the
Per-Arnt-Sim domain superfamily (6, 7). In the ground state the
chromophore is deprotonated and buried in a hydrophobic pocket of the
protein where its negative charge is stabilized via a hydrogen bonding
network. Absorption of light induces a photocycle in E-PYP, in which
isomerization of the chromophore is the initial step, which leads to
the formation of several transient intermediates on the femtosecond to
nanosecond timescale (8, 9). Within a few nanoseconds an
intermediate is formed (pR465, also named I1 or
PYPL;
max = 465 nm) and red-shifted
with respect to the ground state absorption maximum (
max = 446 nm). pR465 decays into a blue-shifted intermediate
(pB355, also named I2 or PYPM;
max = 355 nm) with time constants of 200 µs
and 1.2 ms (10, 11). This latter transition is accompanied by
protonation of the phenolic oxygen of the chromophore and by subsequent
conformational changes of the protein (12, 13). It is suggested that
pB355 is the signaling state of PYP. From pB355
the ground state pG446 is recovered in a biexponential
process with time constants of 200 ms and ~1 s (11). The
pR465 to pB355 and pB355 to
pG446 transitions are very sensitive to both temperature
and pH (14, 15).
In contrast to the detailed knowledge available for PYP from E. halophila, other photoactive yellow proteins are biophysically poorly investigated. So far, proteins from three other species were
purified and basically characterized: (i) PYP from Rhodospirillum salexigens (16), which shares 71% amino acid sequence identity with E-PYP and has virtually the same ground state absorption spectrum
(
max = 445 nm) and similar kinetics of photobleaching and recovery (with time constants for pB formation and pG recovery of
85 µs and 210 ms, respectively); (ii) PYP-phytochrome-related protein from Rhodospirillum centenum (17), a
hybrid-protein, consisting of 884 amino acids, with an N-terminal PYP
domain fused to a central phytochrome-like domain and a C-terminal
histidine kinase domain. When heterologously expressed and
reconstituted with 4-hydroxy-cinnamic acid, Ppr displays an absorbance
maximum at 434 nm and is photoactive; bleaching at 434 nm is
accompanied by the initial formation of a red-shifted intermediate with
a difference absorption maximum at ~470 nm, and subsequently a
blue-shifted intermediate is formed with a difference absorption
maximum at ~330 nm. The recovery to the ground state is biphasic with
a fast and a very slow component (lifetimes of 0.21 ms and 46 s,
respectively); and (iii) PYP from Rhodobacter sphaeroides
(R-PYP), which has been characterized in some more detail (18) and is
also the subject of this study.
Heterologously expressed R-PYP, reconstituted in vitro with
4-hydroxy-cinnamic acid, is a yellow-colored and photoactive protein (18). The main absorption band with a maximum at 446 nm
(R-PYP446) can be reversibly bleached by irradiation with
blue light, which leads to the formation of a blue-shifted intermediate
with a difference absorption maximum at 360 nm (formerly designated as
pB350; because of our new results presented in this paper
it is now named pB360). pB360 of R-PYP relaxes
to the ground state of R-PYP446, pG446, with a
time constant of 2 ms. This recovery process is ~100-fold faster than
in E-PYP and ~23.000-fold faster than in Ppr.
Moreover, the UV-visible absorption spectrum of R-PYP shows an
additional peak, positioned at 360 nm, named R-PYP360.
R-PYP360 and R-PYP446 are jointly part of a
temperature- and pH-dependent equilibrium.
R-PYP360 and R-PYP446 can be reversibly
interconverted by increasing/decreasing the temperature. Lowering the
temperature leads to accumulation of R-PYP360. Titration of
the ground state of R-PYP in the pH range from 1.5 to 9 revealed two
separate transitions, with pKa values of 3.8 and
6.5 (18). Below pH 9 the absorbance at 446 nm decreases, whereas the
absorbance at 360 nm increases at lower pH. Below pH 5, yet another
spectral intermediate is formed, with a clearly further blue-shifted
absorbance maximum (345 nm). This form is probably analogous to
pBdark of E-PYP, which is a partially unfolded protein
state, formed at low pH (pKa = 2.7) (19).
In the present study we extend our analysis of the photoactive
properties of R-PYP. To gain a deeper insight into the photocycle of
R-PYP446 we measured laser-induced transient absorption
changes, with high spectral (charged-coupled device (CCD)
camera) and temporal (photomultiplier) resolution at two
different temperatures. Measurements were complicated by the light
sensitivity of R-PYP360, which also undergoes a photocycle
after absorption of light. In addition, the protein conformation of the
two forms of R-PYP was examined with respect to accessible hydrophobic
surface areas, using the fluorescent polarity probe Nile Red. The
isomerization and protonation state of the chromophore and some
features of the hydrogen bonding network for both ground state species
of R-PYP and their longest living photocycle intermediates were studied
by FT-IR spectroscopy.
 |
MATERIALS AND METHODS |
Sample Preparation--
R-PYP was heterologously overexpressed
in Escherichia coli and purified as described earlier (18).
Apo-R-PYP was reconstituted using activated 4-hydroxy-cinnamic acid as
described previously (14) or 7-hydroxy-coumarin-3-carboxylic acid
(referred to as locked chromophore) as in Ref. 20. E-PYP was produced
and purified as described (1). Samples were analyzed in 50 or 100 mM Tris-HCl buffer at pH 7.5 to 8.
Steady State and Transient UV-visible Measurements--
Steady
state absorption spectra of R-PYP were measured on a HP 8453 UV-visible
diode array spectrophotometer (Hewlett-Packard Nederland BV,
Amstelveen, The Netherlands). The photocycle of R-PYP360
was also investigated on this spectrophotometer using variable time
resolutions ranging from 1 to 300 s. R-PYP360 was excited using a photo flashlight (500-µs pulse width) equipped with a
330 ± 50-nm band-pass filter. To examine spectral changes after a
subsequent blue flash, the same set-up was used but with a 450 ± 7-nm interference filter.
Laser-flash Photolysis Spectroscopy--
The photocycle of
R-PYP446 was studied using an Edinburgh Instruments Ltd.
LP900 spectrometer (Livingston, West Lothian, United Kingdom), equipped
with both a CCD camera and a photomultiplier, in combination with a
Continuum Surelite optical parametric oscillator laser (for further
details see Ref. 21). The sample was excited with 465-nm laser flashes
of 6 to 9 mJ (pulse width 6 ns). In a number of experiments, a 400-nm
long-pass or a 450 ± 7-nm interference filter was introduced into
the observation light beam, before the sample, to reduce secondary
photochemistry. The probe light intensity was maximally reduced, while
maintaining an acceptable signal/noise ratio. Time-gated spectra were
recorded using the CCD camera, averaging 10 to 50 single measurements.
Time traces were measured at different wavelengths between 400 and 500 nm, using the photomultiplier. Data of 64 recordings were averaged for
each trace.
The measurements were carried out at 20 ± 1 or 7 ± 2 °C
in a water-cooled sample cell. The temperature was regularly monitored directly in the sample. For experiments at 7 °C the sample chamber was flushed with nitrogen gas to prevent condense formation. Data were
globally fitted by multiexponential functions using nonlinear least-square procedures from the Microcal Origin software package or
with the help of a home-developed global and target analysis package
described elsewhere (22, 23).
Fluorescence Experiments--
Fluorescence was measured in a
1-cm cuvette using an AMINCO Bowman Series 2 luminescence spectrometer
(Thermo Spectronic, Rochester, NY). For determination of the emission
spectra of the two spectral species of R-PYP, the excitation
wavelengths were 446 and 360 nm (bandwidth, 16 nm), and emission was
recorded at a rate of 1 nm/s from 450 to 600 nm and from 365 to 600 nm
(bandwidth, 4 nm), respectively. Fluorescence excitation spectra were
detected from 300 to 490 nm and 300 to 430 nm (bandwidth, 16 nm) by
measuring the emission at 496 and 440 nm (bandwidth, 4 nm),
respectively. The fluorescence quantum yield for the 446-nm spectral
form of R-PYP in 50 mM Tris-HCl, pH 8, was determined by
comparing its fluorescence with that of E-PYP (
fl = 0.002) (24). Both samples were excited at 446 nm with equal absorption
at this wavelength.
Nile Red Binding Assay--
For the Nile Red binding studies, 20 µl of a 100 µM Nile Red stock solution (in dimethyl
sulfoxide) was added to a 1980-µl sample of R-PYP or locked R-PYP
(R-PYP reconstituted with the locked chromophore) with an
A446 of 0.1. Measurements were started 30 s
after the addition of the probe. The emission spectrum was recorded
from 555 to 800 nm (bandwidth, 4 nm) with excitation at 540 nm
(bandwidth, 16 nm). The measurements were carried out at room
temperature (~20 °C) or at ~12 °C. The cuvette was
water-cooled; the temperature was monitored in the cuvette.
FT-IR Spectroscopy--
FT-IR difference spectroscopy was
performed on a Bruker IFS 66v spectrometer. Spectral resolution was set
to 2 cm
1 in the photoconversion experiments and 4.5 cm
1 in the low temperature experiments. In the
conventional transmission technique, used for the photoconversion
experiments, a droplet of a highly concentrated sample solution was put
on a BaF2 cuvette and sealed by a cover window of the same
material (see Ref. 25 for more experimental details). For light
excitation either the 3rd harmonic of a Nd:YAG laser (Quanta Ray GRC
12S) was used (20 pulses of 8-ns duration and 20 mJ of energy at
355 nm each), or this laser light was fed into a tunable optical
parametric oscillator to produce 100 pulses of 5-ns duration each and 2 mJ of energy at 445 nm. Laser emission was guided to the sample
through a quartz fiber bundle to achieve homogenous illumination.
Low temperature, blue light-induced, FT-IR difference spectroscopy was
performed using the attenuated total reflection technique. An
attenuated total reflection accessory was used with a diamond disc as
the internal reflection element (26). 10 µl of the protein solution
was put on the diamond surface and concentrated by a stream of
nitrogen. Rehydration by 2 µl of water resulted in a 2:1 ratio of the
band heights around 1650 cm
1 (amide I overlapped by
H2O bending mode) and 1550 cm
1 (amide II),
which demonstrates good hydration of the protein. Blue light
illumination was done via a cold light source equipped with a fiber
bundle (Schott, Mainz, Germany). In all light-induced difference
experiments, a broadband interference filter (OCLI) was inserted in
front of the mercury-cadmium-telluride detector to protect it from
stray light and to limit the spectral range to 1850 to 950 cm
1. The temperature of the sample was controlled by a
circulating water bath filled with ethanol.
 |
RESULTS |
Fluorescence Spectra of R-PYP--
The absorption spectrum of
R-PYP is characterized by the presence of an equilibrium between two
species with maxima at 360 and 446 nm, respectively. The fluorescence
emission and excitation spectra of these species are shown in Fig.
1. Excitation at 446 nm yielded an
emission spectrum with a maximum at 496 nm. Excitation at 360 nm
resulted in additional fluorescence at ~440 nm. The fluorescence
quantum yield of R-PYP446 was calculated as 0.03 (see
"Materials and Methods"). The corresponding fluorescence excitation
spectra (Fig. 1), recorded at 440 and 496 nm, yielded maxima at 360 and
446 nm, respectively, representing the two forms, R-PYP360
and R-PYP446, as also observed in the absorption
spectrum.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Fluorescence emission and excitation spectra
of R-PYP. A, emission spectrum with excitation at 446 nm and excitation spectrum monitored at 496 nm are plotted. The
dashed line represents the absorption spectrum of R-PYP
(adapted to scale for convenience). B, emission spectrum
with excitation at 360 nm and excitation spectrum monitored at 440 nm
are plotted.
|
|
Photocycle of R-PYP446--
The ability to undergo a
photocycle after light absorption is a characteristic feature of all
known PYPs. Also the yellow form of R-PYP, R-PYP446,
displays such a photocycle. Absorption difference spectra recorded
after excitation with a 465-nm laser flash are shown in Fig.
2. After 30 ns, positive absorption
changes at 380 nm and at 480 nm are visible, together with a large
negative signal at 446 nm reflecting the bleach of the ground state. At the next time point (500 ns) the positive red-shifted absorption, with
respect to the ground state absorption maximum at 446 nm, has
decreased, whereas the blue-shifted absorption has shifted toward 355 nm. In the following phase the blue-shifted absorption increases,
accompanied by a further decrease in the absorption at 446 nm. These
processes are completed at 60 µs (Fig. 2A). The recovery
phase of the photocycle is shown in Fig. 2B, where the blue-shifted intermediate is returning into the ground state
pG446 within 10 ms. This recovery seems to be incomplete.
We attribute this finding (a residual absorption difference) to
secondary photochemical processes induced by the observation light (see
below).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
Time resolved difference absorption spectra
of R-PYP at 20 °C after 465-nm laser excitation. Spectra
1 to 8 are taken after 30 ns, 500 ns, 5 µs, 15 µs, 60 µs,
500 µs, 1 ms, and 10 ms, respectively.
|
|
Photocycle of R-PYP360--
In addition to
R-PYP446, R-PYP360 can also be bleached by
light. Fig. 3A shows an
absorption difference spectrum of R-PYP after illumination with UV
light. The bleach of R-PYP360 is accompanied by an increase
in absorption at ~435 nm; we have named this latter state
R-PYP435. This transformation occurs within a time shorter than 100 ms. A subsequent 450-nm light flash induces the recovery of
R-PYP360 (not shown), again faster than 100 ms. Both
phototransformations could not be studied at higher time resolution
because of photochemistry induced by the observation light. Obviously
this effect is getting more pronounced at higher probe light
intensities, obligatory for nanosecond to millisecond flash photolysis
experiments. R-PYP435 returns back to R-PYP360
in the dark on a minute time scale (see Fig. 3B).
Determination of a more accurate time constant again is not possible
(in our set-up) because of the extreme light sensitivity of
R-PYP435.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
The photocycle of R-PYP360.
A, absorption difference spectrum of R-PYP after a UV flash
(330 ± 50 nm). B, time course of the absorption at 440 nm (filled symbols) and 360 nm (open symbols)
after a UV flash (applied at the 500-s time point).
|
|
This second photocycle of R-PYP also explains the residual absorption
difference in the laser excitation (465 nm) photocycle measurements
(see Fig. 2B). The observation light below 400 nm photoactivates R-PYP360 and, as a result, leads to
accumulation of the R-PYP435 intermediate (with a minute
lifetime). Subsequently, 465-nm laser light induces, in addition to the
intended photoactivation of R-PYP446, the light-driven
recovery of R-PYP435 to R-PYP360. A
photoequilibrium of R-PYP360 (excited continuously during
the 100-ms measurement cycle by the observation light) and
R-PYP435 (photoconverted within 6 ns by the blue excitation
laser) is then established, which is reflected in a residual absorption
difference (see e.g. the 10-ms absorption difference
spectrum in Fig. 2B).
To determine the influence of this secondary photochemistry on the
characteristics of the R-PYP446 photocycle we performed laser flash photolysis experiments while using a 450 ± 7-nm
band-pass filter placed into the observation light beam. Fig.
4A shows kinetic traces
recorded at 450 nm after excitation with a 465-nm laser flash. The
bleach at 446 nm occurs biexponentially with time constants of 0.5 and
17 µs (see Table I). The recovery of
the ground state can be fitted monoexponentially with a time constant
of 2.8 ms. Time constants determined in the absence or presence of the
filter in the observation light path are virtually identical (see Table I). Recovery, however, is completed to zero when using the 450-nm filter and to a negative value, reflecting R-PYP435
transformed to R-PYP360, without the filter (Fig.
4B).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4.
Kinetic traces of absorbance changes at 450 nm after 465-nm laser excitation at 20 °C. A, traces
were measured in four different time windows. A 450 ± 7-nm
interference filter was introduced in the observation light beam.
B, traces were measured in a time window up to 20 ms. No
filter (open circles) or a 450-nm interference filter
(filled circles) was introduced in the observation light.
Each data point represents the average of five time points.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Time constants measured for the R-PYP446 photocycle
Measurements of kinetic traces at 450 nm were carried out with or
without a 450 ± 7-nm interference filter in the observation light
beam at 20 °C (upper part). Data for measurements at both
temperatures were determined from time-resolved measurements combining
the analysis of time-gated spectra and time-traces (lower part). Traces
were globally fitted for all measured wavelengths from 400 to 500 nm
with two components for the bleach and one component for the recovery
of the 446-nm form. The data from the recorded spectra were averaged
over 9 nm, and a tri-exponential global fit was performed over all
wavelengths and time points.
|
|
The R-PYP446 Photocycle at Two Different
Temperatures--
To examine whether the formation of the different
intermediates is temperature-sensitive, we studied the
R-PYP446 photocycle at two different temperatures (20 and
7 °C). A 400-nm long-pass filter in the observation light beam was
used to prevent secondary photochemistry (see above). The entire
photocycle is slowed down 2- to 3-fold at 7 °C (see Table I). Fig.
5 shows the calculated decay-associated
difference spectra from the data obtained in the time-resolved CCD
measurements, for both temperatures. At 7 °C the shape and relative
amplitude of the first decay-associated difference spectra compared
with the second are distinctly different from the situation at
20 °C. At low temperature the intermediate(s) absorption decay in
the range of 400 to 440 nm is much more associated with the first
component. The varying shape and relative amplitude of the
decay-associated difference spectra indicate that indeed a mixture of
intermediates is formed during the photocycle, of which the relative
amounts vary in a temperature-dependent way.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Decay associated difference spectra of the
R-PYP446 photocycle. CCD spectra were measured at
20 °C (A) and 7 °C (B) with a 400-nm
long-pass filter placed in the observation light beam. Difference
spectra were averaged over 9 nm, and data were globally fitted with a
triexponential function. The amplitude for the first
(triangles), second (squares), and third
(circles) component was plotted.
|
|
Time-resolved spectra analogous to those shown in Fig. 2 were subjected
to global target analysis using a sequential model with three
components, and the fluorescence excitation spectrum were obtained with
detection at 496 nm (see Fig. 1) as the ground state spectrum
pG446 of R-PYP446. To be able to include
information on the absorption changes below 400 nm, we generated data
in time-gated measurements without the placement of a filter in the
observation light beam. The estimated species-associated spectra of the
three components at 20 °C are shown in Fig.
6A. Clearly, the 3rd component (dashed) resembles the typical blue-shifted intermediate pB,
known from E-PYP and Ppr, with a maximum at 360 nm (pB360,
formerly designated as pB350) (18). The spectra of the
first two components are very broad, and the second is also very
structured. They presumably both represent a mixture of several
intermediates, including a species analogous to pR465 from
E-PYP, but obviously there is also at least one with a blue-shifted
absorption maximum (with respect to R-PYP446).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
Species-associated spectra (SAS) of the
R-PYP446 photocycle at 20 °C (A) and
7 °C (B). Spectra for three photocycle
components were calculated from global target analysis of time-resolved
difference spectra after a 465-nm laser flash. The estimated spectra of
the first (solid line), second (dotted line), and
third (dashed line) component are shown. Subsequently, the
spectra of the first two components at both temperatures were globally
fitted to a sum of three gaussian functions (with maxima at 358, 414, and 466 nm and standard deviations of 25, 29, and 16 nm, respectively)
to resolve the spectra of the presumed species (dashed-dotted
line). The relative amplitudes (i.e. the product of
concentration and extinction coefficient) of the three spectra,
pB'360, pB415, and pR465, are
calculated as 27, 59, and 14% (1st SAS, 20 °C), 45, 38, and
17% (2nd SAS, 20 °C), 100, 0, and 0% (3rd SAS, 20 °C), 17, 62, and 21% (1st SAS, 7 °C), 45, 33, and 22% (2nd SAS, 7 °C), and
73, 17, and 0% (3rd SAS, 7 °C).
|
|
The estimated species-associated spectra for the photocycle at 7 °C,
presented in Fig. 6B, again show the formation of a
blue-shifted pB360 intermediate (dashed), now
with some additional absorption at about 410 nm, compared with the
spectra measured at 20 °C. Moreover, again the first and second
species-associated spectra are very broad, the latter showing again
sub-structures, but with less absorption at around 410 nm and a more
pronounced contribution at 465 nm. Comparison of the species-associated
spectra obtained at both temperatures clearly shows the presence of
several components prior to the formation of pB360 in the
R-PYP446 photocycle. We performed a global fit for these
data to resolve the spectra of the involved species (shown also in Fig.
6). For the first two intermediates the presence of three species with
absorption maxima at 360, 415, and 465 nm (pB'360,
pB415, and pR465, respectively) can be fitted
reasonably well. The relative contributions do vary with temperature
(see legend of Fig. 6). Subsequently, pB360 is accumulated
with, at 7 °C, a contribution of the 415 species.
Nile Red Bindings Assays--
In R-PYP we have found the special
situation for PYP that both species visible in the absorption spectrum
are photoactive. To examine the nature of these two forms we have
performed Nile Red (NR) binding assays. NR can be used as a fluorescent
probe to obtain information about the accessible hydrophobic surface area of a protein. The fluorescence of NR in a hydrophilic environment (here aqueous buffer) is very low and has a maximum at 660 nm (Fig.
7). When R-PYP is added, the fluorescence
increases and shifts toward shorter wavelengths (
max = ~620 nm), reflecting the binding of NR to a hydrophobic surface (12).
In contrast, the binding of NR to R-PYP reconstituted with a locked
chromophore (displaying a single absorption band at 441 nm) (18) is
very low, and the fluorescence maximum is only slightly blue-shifted (
max = ~657 nm). Presumably, R-PYP360 is
primarily responsible for binding of NR because of the exposure of a
hydrophobic region. This finding is supported by the change in
fluorescence (both in amplitude (increased) and position (blue-shifted)
of the maximum) at lower temperatures, where because of the thermal
equilibrium between R-PYP360 and R-PYP446, a
transition takes place from R-PYP446 to
R-PYP360 (18). The change in the NR emission is reversible as expected for a thermal equilibrium (results not shown).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Temperature-, pH-, and chromophore-type
dependence of Nile Red binding to R-PYP. From a 100 µM Nile Red stock solution, 20 µl were added to 1980 µl of sample and mixed immediately by inverting. A fluorescence
emission spectrum with excitation at 540 nm was recorded 30 s
after addition of NR. Spectra are shown for NR added to R-PYP at
20 °C, pH 8 (solid line), 12 °C, pH 8 (dotted
line), 20 °C, pH 5 (dashed-dotted line), to
locked R-PYP at 20 °C, pH 8 (dashed-dot-dot line),
and to the buffer at 20 °C, pH 8 (dashed line).
|
|
Another possibility to drive the equilibrium more toward
R-PYP360 is a decrease of the pH. As can be seen from Fig.
7, a change to pH 5 increases the fluorescence dramatically,
demonstrating again the presence of an exposed hydrophobic region. The
maximum of the fluorescence emission is red-shifted (
max = ~640 nm) compared with R-PYP at pH 8, indicating a change in the
characteristics of the Nile Red binding site, possibly because of
protonation of an amino acid in or near the Nile Red binding site.
FT-IR Spectroscopy--
The main focus of the FT-IR spectroscopic
experiments was the determination of the protonation and isomerization
state of the chromophore in both ground state forms of R-PYP. The long lifetime of R-PYP435 allowed us to accumulate
R-PYP435 by excitation with 355 nm of light. The
corresponding FT-IR difference spectrum (R-PYP435-R-PYP360) is shown in Fig.
8 (trace 1).
R-PYP435 could be illuminated back to R-PYP360
by using 445 nm of laser light. In the corresponding difference
spectrum (Fig. 8A, trace 3) all the major bands
appear with a reverted sign. This illumination back and forth was
reproducible with high accuracy. Trace 2 shows the sum of
the two difference spectra (corrected for the difference in the extent
of photoconversion because of our experimental conditions) demonstrating the reversibility. The high noise in trace 2 in some regions (i.e. in the ranges from 1695 to 1570 cm
1 and 1120 to 980 cm
1) because of strong
absorption of the sample indicates that accuracy of band positions and
amplitudes of the difference spectra will be low in these regions.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 8.
FT-IR difference spectra. A,
reversibility of R-PYP360-R-PYP435 infrared
absorption: FT-IR absorption difference spectra
R-PYP435-R-PYP360 (trace 1; after
excitation at 355 nm), R-PYP360-R-PYP435
(trace 3; after subsequent excitation at 445 nm), and the
baseline (trace 2; calculated as the sum of the two
spectra). B, chromophore protonation and isomerization
state: FT-IR difference spectra for
pB355-E-PYP446 (trace 5),
pB360-R-PYP446 (trace 4), and
R-PYP435-R-PYP360 (trace 1). The
indicated bands report about the chromophore protonation and
isomerization state and the carboxylic group of Glu-46. The values for
the band positions are taken from the literature (14, 27, 28).
|
|
A first inspection of the R-PYP435-R-PYP360
difference spectrum (Fig. 8A, trace 1) shows a
number of bands known (and partially assigned) from FT-IR studies on
E-PYP (e.g. the positive bands at 1627, 1489 (with a
shoulder at 1504), 1304, and 1158 cm
1 and the negative
bands 1645, 1516, 1287, and 1170 cm
1). Some other large
features (e.g. at 1145 and 993 cm
1), however,
are new or very different in their intensity. Interestingly, no change
is observed above 1700 cm
1 indicating no change in the
Glu-46 protonation state and the hydrogen bonding strength of its
deproteonated carboxyl group.
R-PYP360 and R-PYP435 have no analogues among
the known intermediate states in E-PYP, though the chromophore and its
binding position are identical, and many important amino acids
(e.g. Glu-46, Tyr-42, Arg-52) are conserved in R-PYP. To use
the assignment of vibrational modes in E-PYP to characterize four R-PYP
states (R-PYP446, pB360, R-PYP360,
R-PYP435) we decided to measure a second difference
spectrum, namely pB360-R-PYP446. When
irradiating a concentrated R-PYP sample at
20 °C with blue
light (445 nm) a photocycle intermediate was accumulated, owing a
lifetime of minutes, that showed a FT-IR difference spectrum
similar to pB355-pG446 of E-PYP (see Fig.
8B, traces 4 and 5, and see
Table II). We tentatively identify
this accumulated intermediate as the photocycle intermediate pB360 from R-PYP446. For the discussion of the
state of chromophore protonation and isomerization, with the help of
assignments obtained in E-PYP, we consider only bands observed in both
difference spectra of R-PYP to exclude false assignments of features
caused by the different amino acid composition and/or different protein
conformation of R-PYP with respect to E-PYP (see Table II). For
comparison we also include a FT-IR difference spectrum
pB355-E-PYP446 (see Fig. 8B,
trace 5, and see Table II). This is very similar to
previously published ones (13, 27, 28) except for the smaller change in
the amide I region because of the relatively low hydration level of the
sample used (29).
View this table:
[in this window]
[in a new window]
|
Table II
Important infrared absorption bands (in cm 1) for
R-PYP446, pB360, R-PYP360, R-PYP435,
E-PYP446, and E-PYP355
|
|
A pair of bands around 1500 cm
1 (the band assigned to the
PYP ground state will be given first from now on), 1498/1515
cm
1 in D2O (13) corresponding to 1485/1515
cm
1 in H2O (27, 28), is attributed to the
phenolic ring vibration of the chromophore (13) and reflects the
protonation state of the chromophore (with the
1515-cm
1 band for the protonated chromophore). Two
spectral features have been assigned to report about the isomerization
state of the chromophore, namely 1302/1286 cm
1 and
1163/~1175 cm
1 (27, 28). A change in the hydrogen
bonding of the carboxyl group of Glu-46 can be monitored from the band
at 1737 cm
1 (1727 cm
1 in D2O)
assigned to the C = O stretching mode of Glu-46 (30).
By comparing the three difference spectra in Fig. 8B, it is
obvious that there are large similarities between
pB355-E-PYP346 and
pB360-R-PYP446, especially at the spectral
features assigned to the protonation and isomerization state of the
chromophore. In contrast, R-PYP360-R-PYP435
shows similar bands but reverted signs. Careful analysis allows us to
determine the protonation and isomerization state of the four R-PYP
species (R-PYP446, pB360, R-PYP360,
R-PYP435; see "Discussion").
 |
DISCUSSION |
Fluorescence--
Fluorescence excitation and emission spectra
were determined for both spectral species of R-PYP. Whereas
R-PYP446 shows a maximum in emission at 496 nm, excitation
at 360 nm reveals an additional fluorescence band centered at 440 nm,
reflecting excitation of R-PYP360. The Stokes shifts for
the emission of R-PYP360 and R-PYP446 are 5051 and 2260 cm
1, respectively. The fluorescence quantum
yield (
fl) for the excitation at 360 nm is much lower
than for excitation at 446 nm (
fl-446).
The fluorescence emission spectrum of R-PYP446 is very
similar to that of E-PYP; the maximum of the latter is only slightly blue-shifted (by 1 nm). Nevertheless, the fluorescence quantum yield
for excitation at 446 nm is increased by about an order of magnitude in
R-PYP (0.03 versus 0.002) (24). Interestingly, the Y42F
mutant E-PYP also shows a highly increased
fl of 0.018 (31). This mutant protein displays also two maxima in the visible part
of the ground state absorption spectrum (391 and 458 nm). However, the
shape of the emission spectrum for excitation at both absorption maxima
remains the same (31), whereas for R-PYP the excitation at 360 nm gives
rise to a clearly different additional emission at ~440 nm.
Excitation spectra recorded at either 440 or 496 nm clearly display the
two species R-PYP360 and R-PYP446.
R-PYP446 Photocycle--
The ability of R-PYP to
undergo a photocycle has been described earlier (18). In this study we
have extended our analyses of the R-PYP446 photocycle to a
broader time range starting from 30 ns up to 20 ms, when the photocycle
is completed. The analysis of time traces measured at a single
wavelength (e.g. at 450 nm; see Fig. 4) yields
three photocycle phases. The bleach of the ground state absorption at
20 °C can be fitted biexponentially with time constants of 0.5 and
17 µs (1.1 and 40 µs at 7 °C), whereas the recovery occurs
monoexponentially with a time constant of 2.5 ms (8 ms at 7 °C).
Although the examination of the R-PYP446 photocycle was
complicated by secondary photochemical events, through absorption of
light by R-PYP360, we were able to show that the two
photocycles do not influence each other. Suppressing the
R-PYP360 photocycle by placing a filter in the observation light beam did not change the time constants for the
R-PYP446 photocycle (see Table I). Global target analysis
of time-gated spectra using a three component sequential model reveals
a quite complex photocycle scheme.
The species-associated spectra (see Fig. 6) for the first two
components at both measured temperatures are very broad and structured;
they possibly represent a mixture of different intermediates. One of
these species absorbing in the red (compared with the ground state
pG446) is similar to pR of E-PYP, with an absorption
maximum at about 465 nm (pR465). The major contribution to
the spectrum of the first component is provided by a species with an
absorption maximum around 415 nm (pB415). The nature of
this species is unclear. The only state of PYP with similar absorption
characteristics reported so far is the low temperature state
PYPBL (32). A third contribution comes from a state with
maximal absorption around 360 nm (pB'360). In the next
species-associated spectrum (after a couple of microseconds at
20 °C) the relative contribution of pB415 has decreased,
but on the other hand the contribution of pB'360 has
increased. Later on, this mixture of presumably three species is
converted with a time constant of 17 µs (20 °C) into a
blue-shifted form with an absorption maximum at 360 nm
(pB360), similar to pB from E-PYP. pB360 and
pB'360 have the same absorption spectra but may differ in
their protein conformation. These two species might also be identical.
A model for the photocycle scheme of R-PYP is shown in Fig.
9. For R-PYP446 (in analogy
with E-PYP) the initial step after light absorption is the
photoisomerization of the chromophore from trans to
cis, leading to the formation of a red-shifted intermediate, pR465. In addition, a species with a protonated chromophore
(
max = ~360 nm), never detected so early in the
photocycles of PYP(-like) proteins, and another intermediate with
so-far unknown characteristics (pB415) are formed. Those
three species exist in a mixture (indicated as
X1 and X2 in Fig. 9),
most probably in a thermal equilibrium (compare the different relative
concentrations at 7 and 20 °C). The blue-shifted absorption spectra
of pB415 and pB'360 point toward a (partly)
protonated chromophore. Such a situation has been described earlier for
the E-PYP mutant Y42F, where a species with absorption at 391 nm can be
observed in the ground state of the protein (31), which is attributed
to a not fully protonated chromophore. Later on in the photocycle the
composition of the mixture, consisting of pR465,
pB415, and pB'360, is changing, and
subsequently pB360 is accumulated with a time constant of 17 µs at 20 °C. pB360 finally returns into the ground
state R-PYP446 with a time constant of 2.5 ms.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 9.
Model of R-PYP including the ground state
equilibrium between R-PYP446 and R-PYP360 and
the two independent photocycles of both forms. Symbols
indicate the isomerization and protonation state of the chromophore and
the conformation of the protein. X1 and
X2 represent a mixture of intermediates formed
in the R-PYP446 photocycle (for details see text).
|
|
The FT-IR difference spectrum pB360-R-PYP446
supports the above stated model at several points. Looking on the
marker bands for protonation and isomerization state it is clear that
the chromophore in R-PYP446 is deprotonated and in
trans conformation, whereas in its photoproduct
pB360 it is protonated and in cis conformation (see Fig. 8 and Table II), a behavior similar to that found for E-PYP446. Furthermore, during the photocycle as for E-PYP,
Glu-46 becomes deprotonated (see below). It is likely that it acts as a
proton donor for the chromophore during the photocycle of E-PYP, as
well as of R-PYP446. In R-PYP446, the negative
charge of the phenolic oxygen of the chromophore is stabilized by
hydrogen bonding to Glu-46. The proton is less strongly bound to
COO
from Glu-46 compared with E-PYP446
(downshift of 10 cm
1). This could reflect a lower
pKa for Glu-46 in the (more) open chromophore
pocket of R-PYP (see below) with respect to that of E-PYP.
The structure of the pB360 form of R-PYP is most probably
different from that of E-PYP, especially in its extent of
conformational changes, which is supposedly lower in the case of R-PYP.
Besides the kinetic argument (2.5- versus 400-ms lifetime
for R-PYP and E-PYP, respectively) there is other support for this
hypothesis. The role of methionine 100 has been extensively studied in
E-PYP (34, 35). It has been suggested that Met-100 facilitates the conformational changes of the chromophore and/or the surrounding amino
acids on the way back to the ground state (35). In all cases,
replacement of the electron donating Met-100 by other amino acids
slowed down the E-PYP recovery reaction significantly (by a factor of
20 to 2000). In R-PYP, Met-100 is replaced by a glycine, but the
recovery kinetics are 100 times faster compared with wild-type E-PYP.
This opposite finding in R-PYP (a much faster recovery) suggests that
the protein conformation and/or the protein environment of the
chromophore in R-PYP are probably different from that in E-PYP.
R-PYP360 Photocycle--
Besides the
R-PYP446 photocycle we also show the ability of
R-PYP360 to undergo a photocycle. After photoexcitation of
R-PYP360 a clear bleach in the absorption around 360 nm is
observed. This bleach is accompanied by the accumulation of a
red-shifted intermediate with an difference absorption maximum around
435 nm (R-PYP435). During this process the chromophore
undergoes a cis to trans isomerization and
deprotonation (see also further below). The ground state
R-PYP360 is recovered via a slow thermal reisomerization
and reprotonation in the dark. Recovery can be accelerated by a
light-induced reisomerization using a subsequent blue flash (450 nm).
Even though there are several examples of (E-)PYP variants described in
the literature, which have two absorption maxima in the ground state
(E-PYP mutants Y42F (31, 36), E46D, and E46A) (37), this is the first
(well described) example of the occurrence of two independent
photocycles in PYP.
The FT-IR difference spectrum R-PYP360-R-PYP435
contains a number of crucial information about the PYP chromophore. The
chromophore of R-PYP360 is deprotonated and in
cis configuration (see Fig. 8 and Table II). On the other
hand, R-PYP435 is protonated and in trans
configuration. Surprisingly, Glu-46 does not change its protonation
state. With respect to other parts of this study, it is likely to
assume that Glu-46 is deprotonated, or at least not hydrogen-bonded to
the chromophore, in R-PYP360. This is a strong argument for
a significant structural difference in R-PYP360 compared
with R-PYP446 and E-PYP446 with respect to the
pKa of Glu-46. Glu-46 behaves much more like a
glutamic acid in solution. Because concomitantly the
pKa of the chromophore is up-shifted by many
orders of magnitudes compared with E-PYP446 and
R-PYP446 it seems justified to propose that its solvent
accessibility in R-PYP360 is much larger compared with
E-PYP446 and R-PYP446.
Despite differences in relative amplitudes, a number of pronounced
infrared absorption bands have been found for R-PYP that are not
present or weak for E-PYP. The most prominent are the bands at 1145 and
993 cm
1. The latter might well be a
hydrogen-out-of-plane mode reflecting a large strain for the
chromophore in R-PYP435. These bands indicate that the
photocycle(s) of R-PYP are different, although similarities might be
seen in the transient absorption changes.
Nile Red--
To obtain information about the conformational state
of both species of R-PYP we used the fluorescent hydrophobicity probe Nile Red (38). This probe was employed recently to examine
conformational changes occurring during the photocycle of E-PYP (12).
It was shown that NR binds to E-PYP upon formation of
pB355, when a hydrophobic region of the protein is exposed.
No binding of NR to E-PYP in the ground state was observed. In
contrast, addition of NR to ground state R-PYP leads to an increase and
a strong blue shift in the fluorescence emission of NR, indicating
binding of NR to an exposed hydrophobic surface. We attribute this
finding to the presence of R-PYP360, because an increase in
the relative concentration of R-PYP360 at lower temperature
leads to a further increase in NR binding. Moreover, results obtained
with apo-R-PYP reconstituted with a trans-locked
chromophore show almost no binding of NR. Note that the interaction of
R-PYP360 with NR is not identical with that of the
signaling state pB355 from E-PYP (12), indicated by the
differences in the maximum of NR emission, 620 and 600 nm, respectively.
R-PYP446 and R-PYP360 are also part of a
pH-dependent equilibrium. The pKa of
this transition was determined as 6.5 (18). The NR emission spectra
after binding to R-PYP recorded at pH 8 and pH 5 differ in their
characteristics. Besides the increase in the amplitude at pH 5, because
of a higher concentration of R-PYP360, the maximum of the
emission has also changed. The red shift in emission can be attributed
to a more polar environment for binding of NR, presumably caused by
protonation of an amino acid near or in the NR binding pocket.
To determine possible NR binding sites, we subjected the model of the
three-dimensional structure of R-PYP (39) to a search for structural
pockets and cavities. Therefore we used the program CastP, which is
publicly available (40) and which provides identification and
measurements of surface-accessible pockets, as well as
interior-inaccessible cavities, for proteins and other molecules. This
analysis revealed the chromophore binding pocket as the largest pocket
in R-PYP. More importantly, the results of the CastP analysis show that the chromophore binding pocket is not buried inside the protein, as is
the case for E-PYP, but that it has direct access to the solvent. This
contact is provided via three mouths, located around the amino acids
Ile-66, Gly-100, and Ala-50 (numbering according to alignment with
E-PYP; amino acids corresponding to Val-66, Met-100, and Thr-50 in
E-PYP). The entries provided via Gly-100 and Ala-50 both lead into a
large tunnel toward the chromophore (as shown in Fig.
10). This tunnel runs along the loop
that connects strands 4 and 5 of the central
-sheet (5) and which
forms the back side of the active site pocket in E-PYP. Met-100 and Thr-50 are key residues in the properties of E-PYP. Both residues shield the chromophore from the hydrophilic environment because of the
presence of a rather large side chain and because of the establishment
of a hydrogen bonding network with Arg-52 for Met-100 and with Tyr-42
and Arg-52 for Thr-50. Mutation of these amino acids lead to severe
changes in the characteristics of E-PYP, which are reflected in the
spectral properties and the photocycle kinetics (34, 41, 42). In R-PYP
natural substitution of these amino acids to Ala-50 and Gly-100 makes
the chromophore binding pocket accessible to the solvent and therefore
is very likely responsible for the observed differences in the
properties of both proteins (see below).

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 10.
3D view of the chromophore binding pocket of
R-PYP. A backbone ribbon structure, with the chromophore
binding pocket superposed, is shown in two views (A and
B). The atoms forming the chromophore binding pocket were
obtained via a CastP analysis (see text) of the modeled structure of
R-PYP (39). The pocket is represented by a solvent contact surface
(1.4-Å probe) with atoms lining the mouth openings in green
and the other atoms in dark gray. The chromophore is shown
as ball and stick in yellow. In panel
A the chromophore can be observed through mouth opening 3. In
panel B the view in panel A was rotated upward to
show the chromophore through mouth opening 1 (the largest of the
three). Mouth opening 2 opens up into the same tunnel to the
chromophore as mouth opening 1. Light green atoms belong to
Ile-66 (mouth 3), Ala-50 (mouth 1), and Gly-100 (shared by mouth 1 and
2). The figure was prepared using the program MOLMOL (33). The program
POV-RayTM (www.povray.org) was used to render the
images.
|
|
Model--
A schematic model describing the different species and
intermediates of R-PYP is depicted in Fig. 9. The two ground state forms of R-PYP characterized by their different absorption maxima (R-PYP446 and R-PYP360) are in a temperature-
and pH-dependent equilibrium (18).
R-PYP446 is characterized by the presence of a deprotonated
trans chromophore (according to the FT-IR results presented
in this paper and in analogy with E-PYP (3, 43, 44)). In contrast, R-PYP360 has a different protein conformation with an
increased accessible hydrophobic surface, as shown from the NR bindings studies. The chromophore of R-PYP360 is protonated, as
already indicated by the strong blue shift in the absorption maximum
and confirmed by results from FT-IR spectroscopy. Additionally, we have
shown by FT-IR spectroscopy that the chromophore in
R-PYP360 is in cis conformation. The latter
conclusion is supported by the following. (i) Apo-R-PYP reconstituted
with a trans-locked chromophore shows only a single peak in
the absorption spectrum, corresponding to R-PYP446;
i.e. R-PYP360 is absent (18). (ii) The different
photocycles of R-PYP446 and R-PYP360 imply a
different conformation of the chromophore in both species. In the
photocycle of R-PYP446 a blue-shifted intermediate
(pB360) is accumulated, characterized by a protonated
trans- to cis-isomerized chromophore (in analogy
with E-PYP) (44). In contrast, the photoactivation of
R-PYP360 gives rise to the formation of a red-shifted
intermediate (R-PYP435), via cis- to
trans-isomerization and deprotonation as indicated by our
FT-IR analysis. R-PYP435 can return to its ground state
(R-PYP360) (including a re-isomerization) either slowly in
the dark or mediated by a 450-nm light flash.
The proposed features for R-PYP446 and R-PYP360
imply a temperature- and pH-dependent equilibrium, where
lowering the temperature or pH induces a transition from
R-PYP446 to R-PYP360 through changes in the
protein conformation, as indicated by the Nile Red binding results,
accompanied by dark isomerization and protonation of the chromophore.
Increasing the temperature or pH triggers the opposite conversion. The
pH dependence, with a pKa of 6.5, can be
explained by the presence of the large chromophore binding pocket that
is accessible to the solvent, as also indicated by the CastP analysis.
Changing the pH of the solvent will have a direct effect on the
protonation state of one or more of the amino acids in the pocket. A
possible candidate for this process is Glu-46, which has a proposed
theoretical pKa value of 6.4 in E-PYP (45).
However, protonation of this amino acid would directly influence the
protonation state of the chromophore and with that the spectral
properties of the protein. A decrease of the pH below 4.5 leads to the
formation of R-PYP345 (analog to pBdark from E-PYP) via unfolding of R-PYP446 and protonation of the chromophore.