Kinetic measurements of the laser induced bacteriorhodopsin photocycle, monitoring changes in the sample absorbance at xed wavelengths of the

visible light
David Sidler April 6, 2012

Experiment performed on March, 20, 2012

1 Introduction
1.1 Theory
Photocycle of bacteriorhodopsin [1] Bacteriorhodopsin is a transmembrane protein, which contains the chromophore retinal, which is essential for the interaction with light. When the retinal molecule in bacteriorhodopsin is excited with light of about 568 nm, a cascade of transitions follows. The absorption of a photon which satises resonance, leads to a conformational change of all-trans retinal to the 13-cis form, thus leading to conformational rearrangement of the surrounding protein. In the activated state, retinal can transfer a proton to Asp 85. Asp 85 hands o the proton to the extracellular space. Retinal is then reprotonated by Asp 96, which receives its proton from the cytoplasm. After this processes, retinal is again in the ground state and the cycle can repeat. For one photon absorbed, one proton is pumped from the cytoplasm to the extracellular space. The whole process is showed shematically in gure 1.1 [2].

Figure 1.1: Scheme of the conformational changes and of retinal in course of the photocycle [2]. Kinetics For the kinetic description of the photocycle, the rst transition bR568 K625 is not taken into account, because it is way to fast to measure with the setup used. It is assumed that within the laser pulse, the maximum amount of K625 is formed. For each other transition, it is assumed to behave like rst order decay of the concerning state, which can be described as: d[A] = k [A] [A]t = [A]0 ekt dt To give more information about the decay in the formula, we set k = 1/ , where is the lifetime. At the lifetime, the initial concentration of the state [A]0 is reduced to [A]0 e1 . The following signal is expected from a single decay K L:

A (K ) [K ] + (L) [L] = (K ) [K ]0 et/ + (L) [K ]0 (1 et/ ) (L) [K ]0 (1 et/ ) [K ]0 et/ + (K ) (L) (L) = [K ]0 + 1 [K ]0 et/ (K ) (K )
Time independent

(L) (K )

[K ]0 et/

A = A et/ When more than one decays occur subsequently, the change of absorbance can be described with a multiexponential function A = y0 + A1 et/1 + A2 et/2 + (1.1)

The number of terms appearing in eqn. 1.1 denote the number of dierent timelifes of decay, between which can be distinguished.
umped npumped Measurement of A For the multiexponential tting, we need A = AP AU . From the Lambert-Beer law it is known, that A = log(I/I0 ), thus P umped I 0 I U npumped I 0 I P umped I U npumped I

A = log

+ log

= log

(1.2)

Since the photodiode is a linear detector light intensities can be substituted by the voltage readout: A = log
P umped U U npumped U

(1.3)

U npumped Baseline ). For U is the voltage measured in DC mode with just the LED turned on (U P umped U , the background has to be substracted from the signal scope. Because the time-resolved signal is measured in AC couple mode, the baseline is supressed and has to be added. Adding this knoledge into eqn. 1.3, we receive Signalscope Background Baseline (U U ) + U Baseline U

A = log

(1.4)

Nanosecond

David Sidler

April 6, 2012

2 Experimental part
2.1 Experimental setup

Figure 2.1: Experimental setup The experimental setup consist of two light beams, a pump beam and a probe beam. The pump beam is a laser, which emitts pulses of light ( 532 nm). By means of a beam splitter, the small part of the beam is led into the trigger diode. The rest of the beam is focussed through the sample with the goal to excite the bacteriorhodopsin molecules. The probe beam is produced by a LED lamp, which has a narrow emission range. The light from the LED is rst collected and then focussed through the sample. The light is again collected and focussed. The LED beam is led through a lter, which should absorb scattered light from the laser source. The focussed light passes a pinhole and arrives at the detector. The detector is a photodiode which measures the incoming light intensity and passes a signal to the oscilloscope. The time is set to zero, when the oscilloscope receives the signal from the triggering photodiode.

2.2 Procedure
For the measurements of the time traces, only four xed wavelengths were measured. The light sources were four dierent LEDs, which provide a narrow band of emitted light. In the following, the LEDs are denoted red ( 642.5 nm), orange ( 592.0 nm), blue ( 470.7 nm) and purple ( 406.0nm). From the UV of the dierent forms of bacteriorhodopsin in the photocycle [3], it was estimated, which processes can be observed at which wavelength. At each measured wavelength except purple, all 4 measured conversions are accompanied by an observable change in the extinction coecient. This means, all 4 conversions can be observed. At the wavelength of the purple LED, only state M4 12 has an extinction coecient which can be distinguished from the other states. Since the transition L550 M412 is much faster than the

decomposition, M412 N520 , only the transitions leading to M412 can be observed. According to this knoledge, the time scales in which the measurements should be carried out, were estimated. They correspond to the timelifes recorded by M.P. Krebs et al. [2]. They are shown in table 2.1. Transition K625 L550 L550 M412 M412 N520 N520 O640 O640 bR568 k [ s 1 ] 7.7E5 2.86E4 286 200 125 [ s] 1.3E-6 3.5E-5 3.5E-3 5.0E-3 8.0E-3

Table 2.1: Calculated lifetimes from literature values [2]. In this timescales were used to measure A

3 Results
3.1 Data 3.2 Data Analysis and Discussion
From resulting data from the time resolved measurements, A(t) was calculated according to eqn. 1.4 for each LED. The dierent timescales were united on one plot. In case of deviations at the overlap, the lower resoluted data was deleted. This led to one graph per LED. To each of this graphs, eqn. 1.1 was tted up to the 4th term. In gures 3.1 to 3.4, the individual ts to the curves received from the four dierent wavelengths are shown. The corresponding parameters are shown in tables 3.1 to 3.4. This parameters were used to initialize the global t, which was done with all four curves. The timelifes were treated as shared parameters. The global t is shown in gure 3.5 and the received timelifes are shown in table 3.5.

Nanosecond

David Sidler

April 6, 2012

2,5x10

-5

2,0x10

-5

1,5x10

-5

OD

1,0x10

-5

5,0x10

-6

0,0

-5,0x10

-6

-1,0x10

-5

10

-7

10

-6

10

-5

10

-4

10

-3

10

-2

10

-1

t [s]

Figure 3.1: Time resolved absorption of red light

Parameter y0 A1 A2 A3 A4 t1 t2 t3 t4

Value -6.757E-7 2.184E-5 9.236E-6 -8.651E-4 8.551E-4 1.141E-6 8.519E-5 4.91E-3 5.01E-3

Standard Error 1.4E-8 4.7E-8 1.9E-8 4.5E-2 4.5E-2 4.1E-9 5.4E-7 3.4E-3 2.2E-3

Table 3.1: Fitted parameters of a multiexponential function to time-dependence of absorption of red light

Nanosecond

David Sidler

April 6, 2012

1,0x10

-5

0,0

-1,0x10

-5

OD

-2,0x10

-5

-3,0x10

-5

-4,0x10

-5

-5,0x10

-5

-6,0x10

-5

10

-7

10

-6

10

-5

10

-4

10

-3

10

-2

10

-1

t [s]

Figure 3.2: Time resolved absorption of orange light

Parameter y0 A1 A2 A3 A4 t1 t2 t3 t4

Value 4.616E-6 3.048E-5 3.236E-5 -1.345E-4 6.873E-5 1.097E-6 5.056E-5 3.90E-3 3.37E-3

Standard Error 3.1E-8 1.3E-7 5.2E-8 4.3E-4 4.3E-4 8.1E-9 2.9E-7 8.7E-4 1.6E-3

Table 3.2: Fitted parameters of a multiexponential function to time-dependence of absorption of orange light

Nanosecond

David Sidler

April 6, 2012

6,0x10

-6

4,0x10

-6

2,0x10

-6

0,0

OD

-2,0x10

-6

-4,0x10

-6

-6,0x10

-6

-8,0x10

-6

-1,0x10

-5

10

-7

10

-6

10

-5

10

-4

10

-3

10

-2

10

-1

t [s]

Figure 3.3: Time resolved absorption of blue light

Parameter y0 A1 A2 A3 A4 t1 t2 t3 t4

Value 4.068E-7 -9.689E-6 1.197E-5 -3.581E-4 3.492E-4 9.015E-7 6.315E-5 2.73E-3 2.65E-3

Standard Error 1.0E-8 6.4E-8 3.0E-8 4.2E-2 4.2E-2 9.7E-9 4.5E-7 3.7E-3 5.3E-3

Table 3.3: Fitted parameters of a multiexponential function to time-dependence of absorption of blue light

Nanosecond

David Sidler

April 6, 2012

2,0x10

-6

1,5x10

-6

1,0x10

-6

OD

5,0x10

-7

0,0

-5,0x10

-7

-1,0x10

-6

10

-5

10

-4

10

-3

10

-2

10

-1

t [s]

Figure 3.4: Time resolved absorption of purple light

Parameter y0 A1 A2 A3 t1 t2 t3

Value -1.336E-7 -4.259E-6 -1.807E-6 2.064E-6 6.399E-7 5.767E-5 4.50E-3

Standard Error 3.5E-9 2.2E-6 1.3E-8 8.1E-9 1.5E-7 1.0E-6 1.0E-5

Table 3.4: Fitted parameters of a multiexponential function to time-dependence of absorption of purple light

Global t

2,4x10 1,6x10 8,0x10

-5

-5

-6

0,0
-6

-8,0x10

OD

-1,6x10 -2,4x10 -3,2x10 -4,0x10 -4,8x10 -5,6x10 -6,4x10

-5

-5

red orange blue purple

-5

-5

-5

-5

-5

10

-8

10

-7

10

-6

10

-5

10

-4

10

-3

10

-2

10

-1

10

t [s]

Figure 3.5: Global t of the multiexponential function to the time-resolved absorption of the four dierent LEDs. The initial values for the amplitudes was taken from the individual ts. The time constants were chosen to be shared variables and were initiated with the literature values.

Parameter t1 t2 t3 t4

Timelifes [s] 1.086E-6 5.304E-5 4.597E-3 4.706E-3

Standard Error [s] 3.7E-9 1.4E-7 1.4E-5 2.9E-5

Lit. Value 1.3E-6 3.5E-5 3.5E-3 8.0E-3

Table 3.5: Lifetimes received from the global t, gure 3.5. Only four timelife constants are found from the multiexponential t. The reason could be, that the transitions M412 N520 and N520 O640 have very similar time lifes like described in [3]. That is why, this two transitions are seen as one with a single time life, which should be bigger than the time life from one single event. The global t matches very good to the experimental data between 107 and 104 s. The major deviation is at the red curve around 103 s. The time lifes received by the measurements agree to the literature values, at least at the order of magnitudes. The reason for this could be, that only 4 terms in the multiexponential function were chosen. Also, the model does not include reversible reactions and side reactions. In addition, the bR solution used for measurements was not newly prepared, which means retinal could have escaped partially from the bR protein and can not undergo the photocycle any more.

10

Nanosecond

David Sidler

April 6, 2012

4 References
[1] W. Khlbrandt, Nature, 2000, 406, 569-570 [2] M. P. Krebs, H. G. Khorana, J. of Bact., 1993, 175, 1555-1560. [3] G. Varo, J. K. Lanyi, Biochem., 1991, 30, 5008-5015.