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            This sample holder was positioned vertical using a supporting base and an additional plate in such a way that the sample holder can be moved up and down via a computer controlled motor to place either the sample cell in the IR beam or the reference cell. This supporting base can also be rotated along the vertical axis to tilt the sample-cell (in this case 45° with respect to the IR radiation) so that the other two beams (a laser light for the bleach and a measuring beam of the fiber-optic spectrometer) also cross at the same area of the sample. The laser beam at 475 nm (30 mW, Roithner Lasertechnik, Austria, diverged further with a lens) was applied at an angle about 90° to bleach the sample. The absorption spectra were measured using optical fiber with UV-Vis-NIR-light source (Mikropack, DT-Mini-2-GS) and CCD detector (avantes, Avaspec-2048). The measuring light of the fibre-optic spectrometer was aligned with an angle about 45° with respect to the sample plate. The temperature of the sample was controlled with a thermostat (Julabo Labortechnik GmbH, Germany) by flowing the water-Glycol mixture across the copper sample holder, which is measured at a position very close to the sample cell using thermo element (Newport Omega).

 

Figure X. The set up of the combined FTIR and UV-Vis spectrometers. The sandwiched PYP sample between two CaF2 plates is mounted in the sample holder, and  placed in the IR radiation in such a way that the other two beams namely from the UV-Vis fiber-optic spectrometer and the bleaching laser also cross at the same area of the sample.

 

The respective FTIR and optical spectrum of the sample were measured either absolute or light minus dark difference, depending on the corresponding spectrum measured either the empty plates or the sample itself of the dark state as a reference

Results

Photocycles of the yellow and intermediate forms, transient absorbance

         The photocycle of the yellow form was measured at pH7 with excitation at 430 nm. Transient absorbance data were taken at 19 wavelengths from 300 to 510 nm. Five of these traces at diagnostic wavelengths are shown in Fig. 1 A. From a global fit of all theses data to a set of exponentials four tim constants

were obtained: t1 = 6 µs, t2 = 100 µs, t3 = 1.3 ms and t4 = 1 s. These are indicated by the dashed vertical lines in Fig 1A. At 100 ns at the beginning of data acquisition there is positive absorbance at 340 and 380 nm suggesting the presence of a species with protonated chromophore. The trace at 500 nm has negligible amplitude indicating the absence of an I1 intermediate on this time scale. The amplitude spectra derived from these data are shown in Fig. 1B. The 6 µs component is of large amplitude and corresponds to a transition from a species absorbing around 380 – 390 nm to the yellow form. We suggest that this transition is due to the relaxation of the ground state equilibrium between the yellow and intermediate forms after depletion of the yellow ground state by the excitation flash at 430 nm. Accordingly the absorbance decreases in the UV and increases around 460 nm. The next two transitions at 100 µs and 1.3 ms are of small amplitude and involve intermediates absorbing in the UV. The amplitude spectra we suggest a blue shift for the 1.3 ms component, which represents the formation of the I2'intermediate. Since the traces for the UV traces in Fig. 1A are of small amplitude and rather noisy, we present in Fig. 1C data at 350 nm obtained with an LED emitting at 350 nm as the light source. This allows data of higher signal to noise ratio and shows more clearly the presence of the µs relaxation followed by two transitions at around 100 µs and 1 ms. The initial bleach of Fig. 1B shows that a species with protonated chromophore absorbing in the UV is formed and confirms the absence of an I1 intermediate absorbing near 470 nm. This initial bleach thus differs in a major way from that of wild type which is characterized by an I1 intermediate in the red and the absence of an intermediate absorbing in the UV (  ).

 

Table 1:

Fluorescence Anisotropy Decay Parameters of Fluorescein bound to Cysteine 140 of Bovine Rhodopsin a

 

b1

b2

b3

r ¥

f1 (ns)

f2(ns)

f3 (ns)

ROS C140-AF

0.18

0.11

 

0.10

0.19

2.9

 

ROS C140-AF bl

0.22

0.08

 

0.09

0.17

2.00

 

OG C140-AF

0.07

0.06

0.22

 

0.10

0.91

30

a Conditions: pH 6.0, 150 mM NaCl, 15 °C

                 

 

The photocycle of the intermediate forms was measured at pH7 with excitation at 355 nm. Data were again taken at 19 wavelengths. The results at the same 5 wavelengths as in Fig. 1A are shown in Fig. 2 B. The data are noisier due to lower fraction cycling of the intermediate form. Apart from the sign of the first µs transition the time traces are strikingly similar to those of the yellow form in Fig. 1A. A global fit of the data at all 19 wavelengths led to four transitions with time constants t1 = 4 µs, t2 = 30 µs, t3 = 1.4 ms and t4 = 1 s indicated by the dashed vertical lines. These time constants are within experimental error the same as those for the cycle of the yellow form. The reversed sign of the amplitude of the first component is consistent with the tentative interpretation of this component in the yellow form.  This time the intermediate form is depleted by the 355 nm flash and the ground state equilibrium shifts from the yellow towards the intermediate forms. Therefore the absorbance should decrease around 460 nm and increase near 380 nm as observed. Moreover, the equilibrium relaxation time should be the same whether the yellow or intermediate form is depleted. The approximate equality of the µs times and the sign reversal of the its amplitude thus strongly support our interpretation. Amplitude spectra are shown in Fig. 2B. The amplitude spectrum for the 4 µs component clearly shows that this is a transition from the yellow ground state to the intermediate form. The 30 µs and 1.4 ms components correspond again to blue shifts indicating the formation of I2'. The initial bleach shows in addition to the expected depletion at 390 nm also depletion at 460 nm. This is due to the fact that excitation at 335 nm also initiates the cycle of the yellow form by absorption in one of its higher excited states. The initial bleach shows that around 100 ns a UV absorbing species is present. This is less clear than in the corresponding initial bleach spectrum of the yellow form due the large negative contribution from the depletion of the intermediate form. Again there is no evidence for an I1 intermediate in the time window from 100 ns to 10 s.