Retinal Binding to Apo-Gloeobacter Rhodopsin. The Role of pH and

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Retinal Binding to Apo-Gloeobacter Rhodopsin. The Role of pH and Retinal-Carotenoid Interaction Sankar Jana, Tamar Eliash, Kwang-Hwan Jung, and Mordechai Sheves J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07523 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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The Journal of Physical Chemistry

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Retinal Binding to Apo-Gloeobacter Rhodopsin. The Role of pH and

2

Retinal-Carotenoid Interaction

3

Sankar Jana †, a, Tamar Eliash†, Kwang-Hwan Jung‡, Mordechai Sheves†*

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†

5

Israel.

6

‡

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Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, South Korea.

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*Corresponding author, E-mail address: [email protected]. Tel: +972-8-

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9344320. Fax: +972-8-9343026.

Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100,

Department of Life Science and Institute of Biological Interfaces, Sogang University,

10 11 12 13 14 15 16 17 18 19 20 21 a

Current address: Department of Chemistry, Graduate School of Science, Tohoku University, 6-3, Aramaki Aza Aoba, Aoba-ku, Sendai 980-8578, Japan Page 1 of 38 ACS Paragon Plus Environment


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Abstract

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Over the last few decades, the structure, functions, properties and molecular mechanisms

3

of retinal proteins have been studied extensively. The newly studied retinal protein

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Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton

5

from the cytoplasmic region to the extracellular region of a cell following light

6

absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In

7

the present study, we report the effect of pH on the binding of retinal to the apo-protein of

8

gR, in the presence and absence of sal, to form the gR pigment. We found that binding at

9

different pH levels reflects the titration of two different protein residues, one at the lower

10

pKa 3.5 and another at the higher pKa 8.4, that affect the pigment's formation. The

11

maximum amount of pigment was formed at pH 5, both with and without the presence of

12

sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190.

13

Furthermore, it is suggested that occupation of the binding site by the retinal

14

chromophore induces protein conformational alterations which in turn effect the

15

carotenoid conformation, which precedes the formation of the retinal-protein covalent

16

bond. Our examination of synthetic retinal analogues in which the ring structure was

17

modified revealed that in the absence of sal, the retinal ring structure affects the rate of

18

pigment formation and that the intact structure is needed for efficient pigment formation.

19

However, the presence of sal abolishes this effect and all-trans retinal and its modified

20

ring analogues bind at a similar rate.

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Keywords: Gloeobacter rhodopsin, salinixanthin, retinal, gR kinetics, retinal protein

2 3

Introduction

4

Retinal proteins, such as bacteriorhodopsin (bR), proteorhodopsin, and

5

xanthorhodopsin (xR), play important roles in transporting protons through the cell

6

membrane following light absorption and transforming energy into an electrochemical

7

gradient. In addition, retinal proteins serve as ion pumps and channels, and act as photo

8

sensors. The mechanism of retinal protein function has been studied extensively and a

9

wide body of knowledge on the molecular level has been obtained

1-12

. It was recently

10

reported that a new retinal protein, gR, was found in the thylacoidless unicellular

11

cyanobacterium Gloeobacter violaceus Pcc 7421,

12

also in E. coli., and 137 of the gR residues are identical (50 %) to those of xR 13. The gR

13

protein acts as a light-driven proton pump, 14 transferring the proton from the cytoplasmic

14

region to the extracellular region of a cell following light absorption.

1, 13

. gR is heterogeneously expressed

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gR is a type I rhodopsin, in which the all-trans retinal chromophore is bound to

16

the Lys-257 amino acid residue of the seventh trans membrane G-helix via a protonated

17

Schiff base formation. Light absorption initiates a photocycle that prompts the

18

deprotonation of the protonated Schiff base, and the protonation of its counter ion triggers

19

proton translocation across the membrane and energy storage in the cell

20

Xanthorhodopsin contains, in addition to the retinal chromophore, a light harvesting

21

carotenoid antenna that efficiently transfers energy to the retinal chromophore. It was

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revealed that the expressed gR protein is also capable of binding sal 13, 17, which transfers

23

at least 40% of its absorbed energy to the retinal chromophore 16.

13, 15, 16

.

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It was reported that the photocycle of gR consists of L-like and N-like states, 14

2

which are converted to the gR initial state through an O intermediate

3

was proposed that the dark state of gR mainly contains an all-trans retinal isomer with a

4

retinal composition that does not change in the light-adapted state. Balashov and

5

coworkers reported the reconstitution of gR with a light-harvesting echinenone and

6

established the role of the 4-keto ring of the carotenoid 17, 18. Demura et al. reported that a

7

trimer or monomer structure of gR is pH-dependent, and that the His-Asp cluster located

8

in the retinal binding site plays a role in controlling the trimer-to-monomer transition 19.

9

Arnold et al. prepared a wide range of gR mutants and established their spectral

10

properties. Among those mutants, one showed a large red-shifted emission with

11

extremely high intensity compared to wild type gR 20. Hegemann et al. performed electro

12

physiological measurements of wild type and mutant gRs and showed that at low and

13

high extracellular pH, the direction of proton translocation is inverted, and that this

14

inversion is responsible for the lower pKa of Glu-132

15

functions as a proton pump in its natural environment and that this proton pumping is

16

compensated by the storage of energy generated by chlorophyll-based photosynthesis

17

without thylakoids 13.

15

. In addition, it

. Jung et al. reported that gR

18

Earlier reports showed that binding of the all-trans retinal to the apo-membrane of

19

bR occurs through several steps. First, the all-trans retinal is rapidly incorporated into the

20

binding pocket, followed by the formation of a red-shifted absorption intermediate called

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the pre-pigment. These events are followed by a slower step in which the retinal-lysine

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protonated Schiff base is formed. Binding of the all-trans retinal to the apo-membrane of


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xR occurs via a similar mechanism, however, in the first step sal changes its

2

conformation when the retinal chromophore occupies the retinal binding pocket 21-23.

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The main goal of the present study was to explore the factors that affect the

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binding of retinal to the apo protein of gR (apo-gR) and the role that sal plays in the

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pigment formation process. We studied the process of all-trans retinal binding to the apo-

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gR at different pH levels and determined the binding rates in the presence and absence of

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sal. We discovered that the binding rate is affected by the pH and that it reflects the

8

titration of two different protein residues, one with a lower pKa of 3.5 and another with a

9

higher pKa of 8.4. The maximum amount of pigment was formed at about pH 5, with and

10

without sal. The presence of sal increased the retinal binding rate by a factor of 190.

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Binding assays involving a series of synthetic retinal analogues modified in their retinal

12

ring and side chain revealed that the intact retinal ring structure is crucial for retinal-sal

13

interactions that affect the retinal binding.

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Experimental section

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Protein preparation and sal extraction and purification

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gR was grown and purified as described previously 14, 16. Sal was extracted from

17

cell membranes of S. ruber. After adding 10 mg/OD sodium dodecyl sulfate (SDS), the

18

mixture was well mixed and lyophilized to remove the water, and then acetone was added

19

to extract the sal, which was purified using a silica gel column. Sal was eluted from the

20

column with 25:75 acetone:n-hexane and, finally, with pure acetone. The ethanol solution

21

containing the all-trans retinal, retinal analogues and sal were added to apo-gR to induce

22

the binding process. The overall ethanol volume was less than 2% of the protein solution



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1

16

2

phosphate/ Tris buffer in 0.06% DDM (n-dodecyl β-D-maltoside).

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. The apo-gR samples were maintained at the required pH using a 50 mM citrate/

3

The apo-gR was prepared by incubating gR with 0.5 M freshly prepared

4

hydroxylamine (pH 7.5) and irradiating it for 20 min with a Schott 250 W cold light

5

source (Carl Zeiss Microscopy, Jena, Germany) equipped with a heat-absorbing filter and

6

an optic fiber (level 4B). The light was filtered through a long pass cutoff filter with a λ

7

of >520 nm (Schott, Mainz, Germany). Next, the sample was filtered through a

8

membrane filter (centricon of 10000 MW) by centrifugation and washed with 0.02%

9

DDM for 5-6 times to remove the retinal oxime and the hydroxylamine that did not react.

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The sample was then stored at 4 °C to avoid reconstitution with all-trans retinal

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originating from residual retinal oxime. The retinal analogues were synthesized as

12

previously described 23-25. Artificial pigments of gR were prepared by incubating the apo-

13

gR (absorbance of 0.1−0.2 OD) overnight with 2 equivalents of the synthetic retinal

14

analogue (1.5 equivalent for all-trans retinal), 50 mM citrate/phosphate/Tris buffer

15

(according to the required pH), and 300 mM NaCl in 0.06% DDM at 25 °C. The

16

concentration of apo-gR (in terms of OD) was determined from the absorption of the

17

pigment at 550 nm during reconstitution with all-trans retinal at pH 7.

18

Uv-Vis spectral measurement

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All the UV-visible spectral measurements were done using an Agilent 4583

20

diode-array spectrophotometer (Agilent Technologies, Palo Alto, CA) equipped with an

21

Agilent 89090A thermostated cuvette holder in a 10 mm quartz cuvette at 25 °C in a dark

22

state. The spectra and kinetic traces of absorbance were recorded after proper background

23

correction with double distilled water. During the kinetic measurements, the absorbance Page 6 of 38 ACS Paragon Plus Environment

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trace was recorded every 60 sec for the first 33 min followed by measurements at 20 min

2

intervals for the remainder of the experiment (i.e., overnight). The processed absorption

3

and difference spectra were generated after internal reference correction (780-800 nm

4

wavelength region was taken as reference where no absorption was detected).

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Absorption kinetics and fitting

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The measured kinetic traces of absorbance at a specific wavelength were

7

normalized and plotted versus time. The kinetic rate constants and fraction ratio were

8

determined form the fitting of traces using the bR second-order growth equation (1) 23.

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y=1-a*exp(-k1*x)-(1-a)*exp(-k2*x)

(1)

10

where, a and (1-a) are fractions of a component with rate constants of k1 and k2,

11

respectively. The fitting of kinetic traces was done in Origin Lab 7. We chose the value

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of a, k1, and k2 depending on the value of the fitting parameter R2 and χ2/dof.

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Determination of pKa of protein residues

14 15

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The amount of pigment obtained at different pH levels was plotted against pH, and the data were fitted to a modified Henderson−Hasselbalch equation (2) 26, 27

F ( x) =

1 1 + 10 n ( pKa − x )

(2)

17

where, n is the number of residues involved in the binding, x is the pH, and pKa is the

18

midpoint of the observed transition.

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Molecular modeling

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To view the three dimensional structure and the arrangement of protein residues

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around the retinal and Lys-257, we used the 3D structure of the gR protein obtained by Page 7 of 38 ACS Paragon Plus Environment


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homology modeling using the Swiss-model server

. As the gR protein has 52.3 %

2

sequence identity and 44 % sequence similarity with xR, we used the crystal structure of

3

xanthorhodopsin (PDB ID 3DLL) 1 as a template for building a structural model for the

4

gR protein.

5

Results

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Our study of the absorption spectroscopy and binding rates of all-trans retinal and

7

several synthetic retinal analogues to apo-gR has shed light on the retinal binding

8

mechanism and retinal-carotenoid interactions, as described below. The absorption

9

maxima of gR pigment and all the artificial gR pigments in their dark-adapted states are

10

summarized in Table 1.

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Binding of all-trans retinal (1) to the apo protein of gR

12

Figure 1a shows the absorption spectral change during incubation of apo-gR with

13

all-trans retinal at different time intervals. The absorption spectra indicate an isosbestic

14

point at 452 nm and a pigment absorption band at 554 nm at pH 5, which reconstituted

15

98% of the pigment. The absorption maximum at 368 nm of the apo-gR is due to retinal

16

oxime 18, 21 and excess all-trans retinal. Figure 1b shows the difference spectra, where the

17

spectrum recorded after 10 sec is subtracted from other spectra. The absorption kinetic

18

traces (Figure 1c) at the pigment maximum obtained from difference spectra (555 nm)

19

indicate biphasic reaction kinetics (k1=4.2 x 10-3 s-1, k2=0.06 x 10-3 s-1; Table 1). The

20

reaction is not monophasic since, as was established for other retinal proteins, all-trans

21

retinal forms the protonated Schiff base linkage with the protein via several steps. First

22

by occupying of the retinal within the binding pocket followed by rearrangements in the


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binding site, formation of the retinal lysine covalent bond and protonation of the Schiff

2

base. 21-23

3

To determine the pH dependency of the reconstitution process, we reconstituted

4

the gR pigment in the 3-9 pH range and plotted the pH vs. the fraction of pigment

5

formation. Figure 2 clearly indicates that within the 5-7 pH range, the pH has a minor

6

effect on pigment formation, but at the lower pH range a sharp decrease in pigment

7

fraction formation is detected. We obtained similar results following a pH increase from

8

7 to 9. Therefore, it is evident that the pigment formation is affected by the protonation

9

state of two protein residues, one with a pKa of 3.5 and the other with a pKa of 8.4

10

(Figure S1a, in supporting information). The pH affects also the reconstitution rate; in pH

11

8.2 (k1=1.7 x 10-3 s-1, k2=0.1 x 10-3 s-1), the rate is 2.5 times slower than in pH 5 (k1=4.2 x

12

10-3 s-1, k2=0.06 x 10-3 s-1) (Table 1).

13

Binding of all-trans retinal to apo-gR in the presence of salinixanthin

14

We examined the reconstitution process and binding rates of all-trans retinal to

15

apo-gR also in the presence of sal. When sal was added to the apo-gR solution, we

16

observed a vibrationally unresolved spectrum, as shown in Figure 3a. After addition of

17

all-trans retinal to the mixture of apo-gR and the sal solution, the absorption maxima

18

(518, 485, and 454 nm) of sal adopted a vibrationally resolved

19

along with increasing intensity. The pigment absorption band appeared around 550-570

20

nm wavelength region. The difference spectra (Figure 3b) indicate that the newly

21

generated absorption band at 565 nm represents the pigment absorption. The rate of

22

pigment formation at the pigment absorption wavelength (565 nm) indicates that the

23

binding process was almost completed within 2 hr (Figure 3c), and that the rate constant

21, 30

, structured band



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k1 is much faster than k2 (k1=0.8 s-1, k2=0.48 x 10-3 s-1; Table 1). The obtained kinetic

2

traces monitoring the change in the sal absorption band at 486 nm produced k1=1.5 s-1

3

and k2=0.20 x 10-3 s-1. It is evident that the sal absorption change was biphasic reflecting

4

the complex binding of the retinal chromophore. The presence of sal accelerated the rate

5

of pigment formation by a factor of 190.

6

Next, we studied the retinal reconstitution process of the apo protein of the gR-

7

salinixanthin (apo-sal) complex within the 3-9 pH range. As shown in Figure 2, the pH

8

dependence curve has a bell shape, with maximum pigment formation occurring at pH 5.

9

It is evident that the presence of sal does not significantly change the bell shape curve

10

relative to retinal binding to the apo protein except for the absence of a plateau region

11

within the pH 5 to 7 region, which was observed in the case of reconstitution without sal

12

(Figure 2). As shown in Figure S1b, the two pKa values of the two protein residues that

13

affected the binding process were at about 2.9 and 8.4. Therefore, the presence of sal did

14

not affect the pKa value in the basic region but lowered the pKa value in the acidic range

15

from 3.5 to 2.9.

16

Binding of synthetic retinal analogues to apo-gR

17

We studied the reconstitution process of different synthetic retinal analogues

18

modified mainly in the ring region (2, 4, 5, 6, 7, 8, 9; Table 1) and in the vicinity of the

19

protonated Schiff base double bond (11, Table 1). In addition, we studied the linear

20

retinal analogue (3, Table 1) and an aromatic retinal analogue characterized by a shorter

21

chain length containing three double bonds (10, Table 1).

22

Reconstitution with retinal analogues modified in the ring region


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The binding of the 3,4-dehydroretinal analogue (2) allowed us to evaluate the

2

effect of an additional double bond in the retinal ring. Figures S2a, b show that

3

incubation of 2 with apo-gR yields a pigment absorption at 580 nm with a biphasic rate

4

process characterized by rate constants of k1=4.1 x 10-3 s-1, k2=0.08 x 10-3 s-1 (Figure

5

S2c, Table 1). The rate is very similar to that observed for all-trans retinal. Reconstitution

6

in the presence of sal produces the structured sal band at 484, 517 nm with a red-shifted

7

pigment band at 585 nm (Figures S3a, b). Analysis of the reaction rate indicated, similar

8

to the native retinal, a biphasic process with k1=0.6 s-1, k2=0.12 x 10-3 s-1 (Figure S3c,

9

Table 1). Analysis of the sal absorption change at 485 nm (k1=1.5 s-1, k2=0.11 x 10-3 s-1)

10

revealed that sal's conformation changes at a faster rate than the process of 3,4-

11

dehydroretinal pigment formation and that the presence of sal enhances the 3,4-

12

dehydroretinal retinal binding rate by a factor of 146 (Table 1).

13

Figure 4a depicts the change in absorption spectra during the binding of linear

14

retinal (3) with the apo-gR at pH 5. The difference spectra (Figure 4b) indicate that the

15

pigment absorption maximum is at 520 nm and that this retinal analogue’s binding

16

occurs at a much slower rate (Figure 4c, k1=0.52 x 10-3 s-1) than the binding of all-trans

17

retinal (Table 1). Reconstitution in the presence of sal at pH 5 produces structured sal

18

bands at 484 and 515 nm accompanied by the appearance of absorption within the 540-

19

560 nm wavelength region (Figure 5a), as further supported by the difference spectra

20

(Figure 5b). Gaussian fitting to resolve the spectra (Figure S4) suggests a pigment

21

absorption band at 550 nm. In this experiment, we also observed a biphasic reaction rate

22

for the pigment formation process (Figure 5c) that was 2115 times faster (k1=1.1 s-1) than



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in the absence of sal (Table 1). We also recorded the kinetics at 484 nm (sal band), where

2

biphasic fitting produced values of k1=1.6 s-1, k2=0.04 x 10-3 s-1 (Table 1).

3

We used the 5-desmethylretinal analogue (4) to elucidate the effect of the 5-

4

methyl group on the retinal binding process. Figure S5a shows the absorption spectral

5

change during incubation with a pigment band at 530 nm (Figure S5b). Fitting of the

6

kinetic traces at 530 nm generated a biphasic reaction rate (Figure S5c, Table 1).

7

Comparison with all-trans retinal indicates that lack of the ring 5-Me group decreases the

8

binding process rate and induces a blue shift of the absorption (25 nm). Reconstitution in

9

the presence of sal developed a red-shifted pigment absorption maximum at 553 nm,

10

which was formed by a biphasic reaction rate, as detected by the pigment formation

11

(Figures S6a-c). Comparison of the rates clearly indicates that sal absorption was altered

12

prior to pigment formation, and that the presence of the sal chromophore accelerates the

13

protonated Schiff base formation process of the 5-desmethylretinal by a factor of 321.

14

Using the 1,1,5-tridemethylretinal analogue (5), which lacks all the retinal ring

15

methyl groups, we evaluated the effect of all the ring methyl groups on the binding of

16

retinal to apo-gR. Figures S7a, b depict the absorption spectral change during incubation,

17

where the pigment band appears at 516 nm (39 nm blue-shifted relative to native retinal)

18

and the binding process exhibits a biphasic rate and slower kinetics than all-trans retinal

19

(Figure S7c, Table 1). Reconstitution in the presence of sal generates a pigment band at

20

545 nm and induces the formation of structured sal bands (Figures S8a, b). The kinetic

21

traces and the fitting parameter at the pigment and sal absorption maxima (Figure S8c,

22

Table 1) suggest that sal undergoes absorption modification at a much faster rate than the


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1

process of 1,1,5-tridemethylretinal pigment formation and, furthermore, that the presence

2

of sal enhances the 1,1,5-tridemethylretinal pigment formation rate by 2444 times.

3

With the 5,6-dihydroretinal analogue (6), we examined the effect of the retinal

4

ring double bond on the binding process of retinal to apo-gR. Figures S9a, b show the

5

absorption spectral change and difference spectra during incubation, where the pigment

6

band appeared at 485 nm. The kinetic traces of the absorption changes

7

biphasic reaction mode (Figure S9c, Table 1). Comparison with all-trans retinal

8

incubation indicated a slower reaction rate in the case of retinal analogue 6. In addition,

9

removal of the C=C ring double bond induces a 70 nm blue-shifted pigment band.

10

Reconstitution in the presence of sal (Figures S10a, b) produces different spectral

11

changes compared to the other retinal analogues. Here, the structured sal bands at 484

12

and 515 nm are formed without detection of a red-shifted pigment absorption band. The

13

pigment's absorption is probably masked by the sal band. The kinetic traces at 484 nm

14

(Figure S10c) produce biphasic rates with k1=1.9 s-1, k2=0.05 x 10-3 s-1 (Table 1). The

15

rates probably represent the formation of both the sal and the pigment bands.

16

Reconstitution with aromatic retinal analogues

indicate a

17

Aromatic methyl retinal (7) contains an aromatic core that substitutes the retinal

18

β-ionone ring. Figures S11a, b depict the absorption spectral change during incubation

19

with apo-gR, indicating a pigment absorption band at 505 nm, formed with a biphasic

20

reaction rate (Figure S11C; Table 1) that is slower than the reconstitution with all-trans

21

retinal. Reconstitution in the presence of sal (Figure S12a) produces structured sal bands

22

with a new red-shifted pigment band at 540 nm (Figures S12a, b). It is evident that the



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1

pigment formation rate (Figure S12c and Table 1) is faster in the presence of sal, and that

2

sal gains its structure faster than the protonated Schiff base formation. The presence of

3

sal enhances the pigment formation rate by a factor of 1100.

4

We also assessed retinal analogue 8, in which the retinal β-ionone ring is

5

substituted by an aromatic -OMe group. Figures S13a, b represent the absorption spectral

6

change during incubation; the pigment forms with an absorption maximum at 521 nm.

7

The rate of absorption change (Figure S13c) indicates that the rate of binding of analogue

8

8 is slower than that of all-trans retinal (Table 1). Reconstitution in the presence of sal

9

(Figures S14a, b) produces a red-shifted pigment absorption band at 550 nm, while sal

10

gains structured bands. The kinetic data (Figure S14c, Table 1) indicate that, in a similar

11

manner to the aromatic methyl retinal (7), sal's absorption change occurs before retinal

12

analogue 8 forms the covalent bond to the protein. Also the presence of sal accelerates

13

the pigment formation rate by a factor of 1309.

14

Additional support for the importance of the intact ionone ring structure for retinal

15

binding was obtained by studying the retinal analogue 9, which contains a dimethyl-

16

amino aromatic core instead of the β-ionone ring. Incubation of this retinal analogue with

17

the apo-gR produces a pigment with a red-shifted absorption at 585 nm (Figures 6a, b).

18

The kinetic traces (Figure 6c, Table 1) clearly show that the binding rate is much slower

19

than that of all-trans retinal and that it is comparable to linear retinal analogue 3.

20

Reconstitution in the presence of sal produced a slightly red-shifted pigment absorption at

21

594 nm (Figures 7a, b) with formation of a fine structured band of sal at 523 nm.

22

Analysis of the reaction rates indicates that the carotene gains its fine structure faster than

23

retinal analogue 9 covalent binding, and that the presence of the carotenoid accelerates Page 14 of 38 ACS Paragon Plus Environment

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1

the retinal analogue 9 binding process by a factor of 1325 compared to the rate in the

2

absence of sal (Figure 7c, Table 1).

3

Retinal analogue 10 represents an aromatic retinal analogue with a shorter

4

polyene chain. The absorption spectral change during the incubation with apo-gR

5

indicates the formation of the pigment at 584 nm with biphasic reaction kinetics (Figures

6

S15a-c, Table 1). The reconstitution experiment in the presence of sal produces a red-

7

shifted pigment band at 591 nm accompanied by structured sal bands (Figures S16a, b).

8

Fitting of the kinetic traces generates biphasic reaction kinetics with k1=1.2 s-1, k2=0.62 x

9

10-3 s-1 (Figure S16c, Table 1). Comparison of the kinetics data indicates that sal gains its

10

structured absorption faster than the pigment formation and that the presence of sal

11

enhances retinal analogue 10 binding by a factor of 1276 (Figure S16c and Table 1).

12

Binding of side chain-substituted retinal analogue

13

The 14-fluoro retinal analogue (11) (Table 1) represents substitution with an

14

electron withdrawing group in the vicinity of the protonated Schiff base region. Figures

15

S17a, b show the absorption spectral change during the incubation process of

16

chromophore 11 with apo-gR and the pigment band formation at 585 nm. Here, too, the

17

kinetic traces (Figure S17c) at the pigment band generate biphasic reaction kinetics, but

18

the k1 (1.3 x 10-3 s-1) rate is slightly slower than that of the all-trans retinal process (Table

19

1). The addition of the electron withdrawing fluorine atom red-shifted the pigment band

20

by 31 nm relative to all-trans retinal. Reconstitution in the presence of sal (Figures S18a,

21

b) produces a structured sal band at 485, 516 nm and a red-shifted pigment absorption at

22

590 nm, which generates biphasic reaction kinetics with k1=0.6 s-1, k2=0.53 x 10-3 s-1

23

(Figure S18c). The kinetic data at 485 nm indicate that sal experiences an absorption Page 15 of 38 ACS Paragon Plus Environment


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Page 16 of 38

1

modification at a rate faster than the 14-fluoro retinal chromophore. In addition, the

2

presence of sal enhances the 14-fluoro retinal binding rate by a factor of 461 (Table 1).

3

3D orientation of protein residues around retinal and Lys-257

4

Our results indicate that, like bR and unlike xR, gR can bind a variety of synthetic

5

retinal analogues both in the presence and absence of sal. Therefore, it appears that the

6

retinal binding site of gR is more flexible than xR. The native retinal length is about 15

7

Å, whereas the longest synthetic retinal studied (retinal analogue 9), which has a length

8

of within 16.5 Å, can easily fit within the retinal pocket, whose length is more than 19 Å

9

(measured from homology model, Figure 8a and b). The 3D model structure around the

10

retinal ring indicates that 20 protein residues are present within the van der Waals

11

distance (Table S1). As shown in Figure 8c, the retinal is surrounded by the aromatic side

12

chain residues Phe-185, Trp-122, Trp-222 Tyr-225 and Tyr-229 (within 4.5 Å). Our

13

comparison of residues around the retinal of gR and xR (Table S1) suggests that both

14

systems share significant similarity. Trp-177 (in the vicinity of the retinal β-ionone ring)

15

is the equivalent of Trp-155 of xR, however, Trp-177 is within 5 Å distance from the

16

retinal ring. Our comparison of residues around Lys (240 in xR and 257 in gR) within 5

17

Å distance indicates that Pro-258 is present in gR, but not in xR. In addition, Phe-260 in

18

gR substitutes Tyr-243 in xR, which may induce a hydrogen bonding alteration. Figure

19

8d depicts several important negatively charged and polar residues (Asp-121, Glu-132,

20

Asp-253, His-87) around the Lys-257.

21

Discussion

22

Among the retinal proteins, gR has maximum sequence similarity with xR (44%)

23

and comparatively less similarity with bR (32%). Similarly to other retinal proteins, we Page 16 of 38 ACS Paragon Plus Environment

Page 17 of 38

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1

assume that apo-gR binds all-trans retinal and synthetic retinal analogues through the

2

formation of a protonated Schiff base entailing at least two steps. First, retinal occupies

3

the binding pocket, followed by the formation of a retinal-lysine covalent bond and

4

protonation of the Schiff base, as reported for xR and bR

5

variety of synthetic retinal analogues modified at the retinal ring and in its polyene chain.

6

In contrast, binding of retinal analogues to apo-xR is much more limited 21, 23. Therefore,

7

it is conceivable that the size of the retinal binding site and the interactions with the

8

protein residues in the vicinity of the retinal are more favorable in gR than in xR, while

9

more similar to bR. The homology model suggests that the amino acids which are close

10

to the retinal are quite similar in gR and xR. Two differences are clearly observed. The

11

presence of Trp-177 within 5 Å distance of the retinal ring region in gR is lacking in xR,

12

and the presence of proline-259 in the vicinity of Lys-257 which forms the protonated

13

Schiff base with the retinal. It is not clear at this stage if these differences induce a major

14

difference between the binding sites of gR and xR. However, the binding of longer and

15

shorter length retinal analogues, like 7, 8, 9 and 10 (Table 1) respectively, indicates that

16

the space availability within the retinal binding site, and/or its flexibility, allow the

17

induction of the necessary protein conformational alteration for the formation of the

18

retinal-protein covalent bond.

21-23

. Apo-gR can bind a wide

19

The present results suggest that pH affects both the percentage of formed pigment

20

and its formation rate following incubation of apo-gR with all-trans retinal in the

21

presence or absence of sal. We have identified two protein residues that affect the

22

process, one with a pKa of 3.5 and the other, 8.4. We propose that, in the absence of sal,

23

the lower pKa is associated with the protonation of Asp-121 residue, whereas, the higher Page 17 of 38 ACS Paragon Plus Environment


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Page 18 of 38

1

pKa is due to the deprotonation of His-87 residue (Figure S1a). The low pKa of Asp-121

2

is probably due to the existence of hydrogen bonds with nearby residues and the close

3

positively charged protonated Schiff base (Figure 8d). Demura et al. reported that the

4

His87-Asp121 cluster also plays a vital role in the monomer-to-trimer transition, which

5

depends on the pH level

6

lower than 3.8. A lower pKa (2.5) of Asp-85 is also reported in the case of

7

bacteriorhodopsin 15, 31, 32. Demura et al. proposed that the pKa of His-87 in gR is 9 19. In

8

the presence of sal, the higher pKa value (attributed to His-87) almost does not change

9

(8.4), but the lower pKa value (Asp-121) somewhat shifts to a lower value (2.9), which

10

19

. Hegemann et al. reported that in gR, the pKa of Asp-121 is

may be due to alteration of the hydrogen bonding network 33.

11

The last step of the retinal binding to apo-gR, the formation of protonated Schiff

12

base covalent bond with the side chain amino group of Lys-257, is formed by a

13

nucleophilic attack of the amino group on the retinal carbonyl. Therefore, the amino

14

group should be deprotonated to achieve an efficient reaction. In water solution, the pKa

15

of lysine is above 10, but it was reported that the pKa value of deeply buried lysine in

16

proteins shifts to a lower pH from 9.2 to 5.3

17

5.8 26. The shifting of pKa to a lower value in apo-gR is probably possible due to: (i) the

18

presence of the counter ion Asp-121 and its proton acceptor capacity; (ii) the position of

19

lysine within the hydrophobic environment; and (iii) a network of hydrogen bonding with

20

protein residues that stabilize the neutral form of Asp-121. Consequently, in pH 5, the

21

maximum amount of free neutral side chain –NH2 groups can act as a nucleophile for the

22

reaction with the retinal to form the Schiff base bond. Similar to bR, pigment formation

23

occurs in gR at pH 3.1 26, probably due to partial deprotonation of the side chain –NH3+

34

; in the case of halorhodopsin it shifts to


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26

1

group of Lys-257

. On the other hand, at higher pH values (>8), the rate of pigment

2

formation is slow and the amount of pigment decreases. It is possible that at high pH,

3

deprotonation of His-87, which closely interacts with Asp-121 and Lys-257, may affect

4

the pKa of Lys-257. Removal of a positive charge in the vicinity of Lys-257 may increase

5

its pKa and prevent Schiff base bond formation.

6

The presented data in Table 1 clearly demonstrate that the binding of apo-gR to

7

synthetic retinal analogues modified in the retinal ring is slower than the binding of all-

8

trans retinal. Therefore, the intact structure of the retinal ring is important for efficient

9

pigment formation. 5-desmethyl retinal (4), which has only two ring methyl groups, binds

10

1.5 times slower than all-trans retinal, whereas retinal analogue 5, which lacks all the

11

methyl groups, binds by one order of magnitude slower. The linear retinal 3, which lacks

12

part of the ring and its methyl groups, binds with a rate comparable to retinal analogue 5.

13

Interestingly, 5,6-dihydroretinal (6; lacks the ring double bond) forms the pigment at one

14

order of magnitude slower than all-trans retinal. The cyclohexane ring of 5,6-

15

dihydroretinal adopts a different conformation relative to native retinal, implying that the

16

conformation of the retinal ring and its intact methyl substituents are crucial for achieving

17

efficient retinal-protein covalent bonding. We propose that the retinal chromophore

18

occupying the protein binding site triggers a protein conformation alteration that is

19

necessary for the formation of the covalent bond. This conformation alteration is

20

efficiently triggered only when the retinal ring has its intact structure. It is well known

21

that the retinal chromophore adopts s-trans ring-chain planar conformation in the binding

22

sites of microbial retinal proteins including gR

23

solution adopts a twisted s-cis ring-chain conformation. Therefore, during the retinal

1, 23, 35

. In contrast, the free retinal in



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Page 20 of 38

1

binding process the retinal chromophore experiences ring-chain planarization process

2

yielding s-trans planar conformation. It is possible that planarization of the retinal ring-

3

polyene chain is necessary to allow formation of the protonated Schiff base double bond

4

between the retinal and apo-gR. The planarization of the retinal may be affected by the

5

ring structure and the intact ring structure is needed for efficient planarization. This

6

proposed mechanism may gain support from our results concerning the aromatic retinal

7

analogues. Modification of the retinal ring by an aromatic ring with a substitution (like -

8

Me, -OMe, -NMe2) decreases the retinal binding rate by a factor of 4 compared to the all-

9

trans retinal. The aromatic retinal analogues adopt a ring-chain planar conformation but

10

still lack the retinal methyl groups. This may explain why their binding is slower but still

11

faster than analogues 3-6. On the other hand, substitution of the side chain near the

12

protonated Schiff base double bond (14-fluoro retinal, 11) caused slower binding relative

13

to native retinal but faster binding than the other analogues modified in the ring. The

14

fluoro substituent may introduce steric hindrance and/or electron distribution alteration

15

that may slow the covalent bond formation.

16

Our findings clearly demonstrate that the presence of sal accelerates the retinal-protein

17

covalent bond formation. Once occupying the retinal binding site, all-trans retinal, as well

18

as the studied synthetic retinal analogues, induce absorption change of the sal as evident

19

by its increased intensity and narrowing the vibronic bands. Similar change was

20

attributed previously, for both xanthorodopsin and gR,

21

relative to the plane of the carotenoid polyene π-system chain, and immobilization of sal

22

in its binding site. Therefore, it is conceivable to propose that occupation of the binding

23

site by the retinal induces protein conformational alteration that allosterically modulates

1, 2, 17

the rotation of the sal ring


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1

the sal conformation and immobilization in its binding site. Moreover, the rate of sal

2

immobilization is almost not affected by the structure of the retinal analogues which

3

probably indicates that highly specific retinal-protein interactions are not needed for the

4

fixation of sal. Interestingly, a higher fixation rate k1 (2.2 s-1) value was detected for the

5

shorter chain length aromatic retinal 10 which may be due to less steric hindrance

6

introduced by this retinal analogue. The sal immobilization precedes the retinal-protein

7

covalent bond, and accelerates this process. We propose that immobilization of sal

8

induces further protein conformational change that reduces the energy barrier for retinal

9

planarization thereby accelerates the covalent bond formation process. This allosteric

10

modulation may have relevance to seven helix receptors and particularly to the family of

11

G protein coupled receptors in which binding of ligands induces bioactivity. The

12

molecular mechanism of this function is not entirely clear and may involve protein

13

conformation alterations triggered by the ligand binding. Our present results demonstrate

14

that sal binding and immobilization modulate the retinal-protein reaction and accelerate

15

it. The results call for further studies to understand the exact mechanism by which the sal

16

accelerates this chemical reaction.

17

18

Acknowledgement

19

This work was supported by the Kimmelman Center for Biomolecular Structure

20

and Assembly, and the Benoziyo Endowment Fund for the Advancement of Science and

21

the J & R Center for Scientific Research. M.S. holds the Katzir-Makineni Chair in

22

Chemistry.



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Page 22 of 38

1

Abbreviation

2

gR: Gloeobacter rhodopsin, apo-gR/apo: Apo protein of gR, sal: salinixanthin, xR:

3

xanthorhodopsin, bR: bacteriorhodopsin, DDM: n-dodecyl β-D-maltoside, Ni-NTA:

4

nickel-nitrilotriacetic acid. SDS: Sodium dodecyl sulfate.

5

Supporting Information

6

Supporting Information contains chemicals, solvents and instruments used; determination

7

of pKa of protein residues in the presence and absence of sal; absorption spectral change

8

and binding kinetics of retinal analogues 2, 4, 5, 6, 7, 8, 10, 11 to apo-gR in the presence

9

and absence of sal; Gaussian fitting of the final difference spectrum obtained from

10

reconstitution of retinal analogue 3 with apo-gR in the presence of sal; list of protein

11

residues in the vicinity of retinal and Lys-257 in gR and Lys-240 in xR within 5 Å.

12

13

14

15

16

17

18

19

20 Page 22 of 38 ACS Paragon Plus Environment

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References 1. Luecke, H.; Schobert, B.; Stagno, J.; Imasheva, E. S.; Wang, J. M.; Balashov, S. P.; Lanyi, J. K. Crystallographic Structure of Xanthorhodopsin, the Light-Driven Proton Pump with a Dual Chromophore. P. Natl. Acad. Sci. 2008, 105, 16561-16565. 2. Balashov, S. P.; Imasheva, E. S.; Boichenko, V. A.; Anton, J.; Wang, J. M.; Lanyi, J. K. Xanthorhodopsin: A Proton Pump with a Light-Harvesting Carotenoid Antenna. Science 2005, 309, 2061-2064. 3. Friedman, N.; Ottolenghi, M.; Sheves, M. Heterogeneity Effects in the Binding of All-Trans Retinal to Bacterio-Opsin. Biochemistry 2003, 42, 11281-11288. 4. Sheves, M.; Albeck, A.; Friedman, N.; Ottolenghi, M. Controlling the Pka of the Bacteriorhodopsin Schiff-Base by Use of Artificial Retinal Analogs. P. Natl. Acad. Sci. 1986, 83, 3262-3266. 5. Slamovits, C. H.; Okamoto, N.; Burri, L.; James, E. R.; Keeling, P. J. A Bacterial Proteorhodopsin Proton Pump in Marine Eukaryotes. Nat. Commun. 2011, 2, 183. 6. Beja, O.; Spudich, E. N.; Spudich, J. L.; Leclerc, M.; DeLong, E. F. Proteorhodopsin Phototrophy in the Ocean. Nature 2001, 411, 786-789. 7. Béjà, O.; Aravind, L.; Koonin, E. V.; Suzuki, M. T.; Hadd, A.; Nguyen, L. P.; Jovanovich, S. B.; Gates, C. M.; Feldman, R. A.; Spudich, J. L., et al. Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea. Science 2000, 289, 1902-1906. 8. Kouyama, T.; Kanada, S.; Takeguchi, Y.; Narusawa, A.; Murakami, M.; Ihara, K. Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas Pharaonis. J. Mol. Biol. 2010, 396, 564-579. 9. Schobert, B.; Lanyi, J. K. Halorhodopsin Is a Light-Driven Chloride Pump. J. Biol. Chem. 1982, 257, 10306-10313. 10. Gautier, A.; Mott, H. R.; Bostock, M. J.; Kirkpatrick, J. P.; Nietlispach, D. Structure Determination of the Seven-Helix Transmembrane Receptor Sensory Rhodopsin Ii by Solution Nmr Spectroscopy. Nat. Struct. Mol. Biol. 2010, 17, 768-774. 11. Gordeliy, V. I.; Labahn, J.; Moukhametzianov, R.; Efremov, R.; Granzin, J.; Schlesinger, R.; Buldt, G.; Savopol, T.; Scheidig, A. J.; Klare, J. P., et al. Molecular Basis of Transmembrane Signalling by Sensory Rhodopsin Ii-Transducer Complex. Nature 2002, 419, 484-487. 12. Luecke, H.; Schobert, B.; Lanyi, J. K.; Spudich, E. N.; Spudich, J. L. Crystal Structure of Sensory Rhodopsin Ii at 2.4 Angstroms: Insights into Color Tuning and Transducer Interaction. Science 2001, 293, 1499-1503. 13. Choi, A. R.; Shi, L.; Brown, L. S.; Jung, K. H. Cyanobacterial Light-Driven Proton Pump, Gloeobacter Rhodopsin: Complementarity between Rhodopsin-Based Energy Production and Photosynthesis. PloS one 2014, 9, e110643. 14. Miranda, M. R.; Choi, A. R.; Shi, L.; Bezerra, A. G., Jr.; Jung, K. H.; Brown, L. S. The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin. Biophys. J. 2009, 96, 1471-1481. 15. Vogt, A.; Wietek, J.; Hegemann, P. Gloeobacter Rhodopsin, Limitation of Proton Pumping at High Electrochemical Load. Biophys. J. 2013, 105, 2055-2063. 16. Iyer, E. S. S.; Gdor, I.; Eliash, T.; Sheves, M.; Ruhman, S. Efficient Femtosecond Energy Transfer from Carotenoid to Retinal in Gloeobacter Rhodopsin–Salinixanthin Complex. J. Phys. Chem. B 2015, 119, 2345-2349. 17. Imasheva, E. S.; Balashov, S. P.; Choi, A. R.; Jung, K. H.; Lanyi, J. K. Reconstitution of Gloeobacter Violaceus Rhodopsin with a Light-Harvesting Carotenoid Antenna. Biochemistry 2009, 48, 10948-10955. Page 23 of 38 ACS Paragon Plus Environment


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18. Balashov, S. P.; Imasheva, E. S.; Choi, A. R.; Jung, K. H.; Liaaen-Jensen, S.; Lanyi, J. K. Reconstitution of Gloeobacter Rhodopsin with Echinenone: Role of the 4-Keto Group. Biochemistry 2010, 49, 9792-9799. 19. Tsukamoto, T.; Kikukawa, T.; Kurata, T.; Jung, K. H.; Kamo, N.; Demura, M. Salt Bridge in the Conserved His-Asp Cluster in Gloeobacter Rhodopsin Contributes to Trimer Formation. Febs. Lett. 2013, 587, 322-327. 20. Engqvist, M. K. M.; McIsaac, R. S.; Dollinger, P.; Flytzanis, N. C.; Abrams, M.; Schor, S.; Arnold, F. H. Directed Evolution of Gloeobacter Violaceus Rhodopsin Spectral Properties. J. Mol. Biol. 2015, 427, 205-220. 21. Imasheva, E. S.; Balashov, S. P.; Wang, J. M.; Smolensky, E.; Sheves, M.; Lanyi, J. K. Chromophore Interaction in Xanthorhodopsin - Retinal Dependence of Salinixanthin Binding. Photochem. Photobiol. 2008, 84, 977-984. 22. Schreckenbach, T.; Walckhoff, B.; Oesterhelt, D. Studies on the Retinal-Protein Interaction in Bacteriorhodopsin. Eur. J. Biochem. 1977, 76, 499-511. 23. Koganov, E. S.; Hirshfeld, A.; Sheves, M. Retinal Beta-Ionone Ring-Salinixanthin Interactions in Xanthorhodopsin: A Study Using Artificial Pigments. Biochemistry 2013, 52, 1290-1301. 24. Nakanishi, K.; Crouch, R. Application of Artificial Pigments to Structure Determination and Study of Photoinduced Transformations of Retinal Proteins. Isr. J. Chem. 1995, 35, 253-272. 25. Sheves, M.; Friedman, N.; Albeck, A.; Ottolenghi, M. Primary Photochemical Event in Bacteriorhodopsin: Study with Artificial Pigments. Biochemistry 1985, 24, 1260-1265. 26. Maiti, T. K.; Engelhard, M.; Sheves, M. Retinal-Protein Interactions in Halorhodopsin from Natronomonas Pharaonis: Binding and Retinal Thermal Isomerization Catalysis. J. Mol. Biol. 2009, 394, 472-484. 27. Rousso, I.; Friedman, N.; Sheves, M.; Ottolenghi, M. Pk(a) of the Protonated Schiff-Base and Aspartic-85 in the Bacteriorhodopsin Binding-Site Is Controlled by a Specific Geometry between the 2 Residues. Biochemistry 1995, 34, 12059-12065. 28. Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Cassarino, T. G.; Bertoni, M.; Bordoli, L., et al. Swiss-Model: Modelling Protein Tertiary and Quaternary Structure Using Evolutionary Information. Nucleic Acids Res. 2014, 42, W252-W258. 29. Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The Swiss-Model Workspace: A Web-Based Environment for Protein Structure Homology Modelling. Bioinformatics 2006, 22, 195-201. 30. Koganov, E. S.; Brumfeld, V.; Friedman, N.; Sheves, M. Origin of Circular Dichroism of Xanthorhodopsin. A Study with Artificial Pigments. J. Phys. Chem. B 2015, 119, 456-464. 31. Lörinczi, É.; Verhoefen, M.-K.; Wachtveitl, J.; Woerner, A. C.; Glaubitz, C.; Engelhard, M.; Bamberg, E.; Friedrich, T. Voltage- and Ph-Dependent Changes in Vectoriality of Photocurrents Mediated by Wild-Type and Mutant Proteorhodopsins Upon Expression in Xenopus Oocytes. J. Mol. Biol. 2009, 393, 320-341. 32. Friedrich, T.; Geibel, S.; Kalmbach, R.; Chizhov, I.; Ataka, K.; Heberle, J.; Engelhard, M.; Bamberg, E. Proteorhodopsin Is a Light-Driven Proton Pump with Variable Vectoriality. J. Mol. Biol. 2002, 321, 821-838. 33. Gat, Y.; Sheves, M. A Mechanism for Controlling the Pka of the Retinal Protonated Schiff Base in Retinal Proteins. A Study with Model Compounds. J. Am. Chem.Soc. 1993, 115, 37723773. 34. Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; García-Moreno E., B. Large Shifts in Pka Values of Lysine Residues Buried inside a Protein. P. Natl. Acad. Sci. 2011, 108, 5260-5265. 35. Van der Steen, R.; Biesheuvel, P. L.; Mathies, R. A.; Lugtenburg, J. Retinal Analogs with Locked 6-7 Conformations Show That Bacteriorhodopsin Requires the 6-S-Trans Conformation of the Chromophore. J. Am. Chem.Soc.1986, 108, 6410-6411. Page 24 of 38 ACS Paragon Plus Environment

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1 2


Table 1: Kinetic data for reconstitution of apo-gR with all-trans retinal and synthetic retinal analogues in the presence and absence of sal at pH 5 apo-gR

Retinal No.

apo-gR + sal b

Structure of retinal analogue

a

pigment band, (k1, k2)x103

Pigment band, k1, k2 x103

carotene peak, k1, k2 x103

r1=k1sal/k1 c r2=k2sal/k2

1

CHO

(555) 4.2(0.86) 0.06(0.14)

(565) 0.8(0.91) 0.48(0.09)

(486) 1.5(0.95) 0.20(0.05)

190 8.0

2

CHO

(580) 4.1(0.83) 0.08(0.17)

(585) 0.6(0.75) 0.12(0.25)

(485) 1.5(0.95) 0.11(0.05)

146 1.5

3

CHO

(520) 0.52(0.34) 0.04(0.66)

(550) 1.1(0.78) 0.03(0.22)

(484) 1.6(0.84) 0.04(0.16)

2115 0.8

4

CHO

(530) 2.8(0.70) 0.04(0.30)

(553) 0.90(0.87) 0.08(0.13)

(484) 1.6(0.92) 0.06(0.08)

321 2.0

5

CHO

(516) 0.45(0.55) 0.06(0.45)

(545) 1.1(0.88) 0.24(0.12)

(484) 1.6(0.88) 0.12(0.12)

2444 4.0

6

CHO

(485) 0.38(0.22) 0.04(0.78)

(484) 1.9(0.81) 0.05(0.19)

-

-

(506) 1.0(0.92) 0.03(0.08)

(540) 1.1(0.88) 0.42(0.12)

(485) 1.4(0.88) 0.38(0.12)

1100 14

(521) 0.84(0.49) 0.08(0.51)

(550) 1.1(0.79) 0.15(0.21)

(485) 1.6(0.87) 0.18(0.13)

1309 1.9

(585) 0.83(0.41) 0.16(0.59)

(594) 1.1(0.29) 0.24(0.71)

(523) 1.6(0.92) 0.18(0.08)

1325 1.5

(584) 0.94(0.46) 0.24(0.54)

(591) 1.2(0.50) 0.62(0.50)

(485) 2.2(0.97) 0.27(0.03)

1276 2.6

(586) 1.3(0.88) 0.06(0.12)

(590) 0.6(0.56) 0.53(0.44)

(485) 1.7(0.94) 0.55(0.06)

461 8.8

CHO

7

8

9

10

CHO O

CHO N

CHO N

CHO

11 F



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

1

a

2

k1 and k2 are the rate constant value in s-1 unit.

3

b

4

presence and absence of sal, respectively. Values within parenthesis represent the

5

contribution of each component.

6

c

7

presence and absence of sal, respectively. Values within parenthesis represent the

8

contribution of each component.

pigment band is the pigment absorption maximum presented in parenthesis in nm unit,

r1 is the ratio of rate constant values (k1) at the pigment absorption maximum in the

r2 is the ratio of rate constant values (k2) at the pigment absorption maximum in the

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23


Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Figure caption

2

Figure 1. (a) Absorption spectra of all-trans retinal incubation with apo-gR. 1: absorption

3

spectra of apo-gR in 0.06 % DDM containing a 50 mM citrate buffer at pH 5 and 300

4

mM NaCl. 2→6: Spectra at different time intervals after the addition of all-trans retinal.

5

(b) Difference spectra; spectra obtained after 10 sec is subtracted from each spectrum. (c)

6

Kinetic traces of absorbance during reconstitution monitored at 555 nm; solid line

7

represents fitted line.

8

Figure 2. Fraction of reconstitution (as monitored by absorption spectrum) of all-trans

9

retinal with apo-gR at different pH values.

10

Figure 3. (a) Incubation of all-trans retinal with apo-gR in the presence of sal, monitored

11

by absorption spectra. 1: Absorption spectrum of apo-gR in 0.06 % DDM containing a 50

12

mM citrate buffer at pH 5 and 300 mM NaCl. 2: Absorption spectra after the addition of

13

sal. 3→6: Absorption spectra at different time intervals after the addition of all-trans

14

retinal. (b) Difference spectra; spectrum obtained after 10 sec is subtracted from each

15

spectrum. (c) Kinetic traces of absorbance during reconstitution at 565 nm; solid line

16

represents fitted line.

17

Figure 4. (a) Monitoring the reconstitution process of linear retinal 3. 1: Absorption

18

spectrum of apo-gR in 0.06 % DDM containing a 50 mM citrate buffer at pH 5 and 300

19

mM NaCl. 2→6: Absorption spectra at different time intervals. (b) Difference spectra;

20

spectra obtained after 10 sec is subtracted from each spectrum. (c) Rate of the

21

reconstitution process detected by absorption at 520 nm; solid line represents the fitted

22

line.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

1

Figure 5. (a) Reconstitution process of linear retinal 3 in the presence of sal. 1:

2

Absorption spectrum of apo-gR in 0.06 % DDM containing a 50 mM citrate buffer at pH

3

5 and 300 mM NaCl. 2: Absorption spectra after the addition of sal. 3→6: Absorption

4

spectra at different time intervals. (b) Difference spectra; spectrum obtained after 10 sec

5

is subtracted from each spectrum. (c) The rate of the reconstitution process was

6

monitored at 550 nm; the solid line represents the fitted line.

7

Figure 6. (a) The incubation process of retinal analogue 9 with apo-gR. 1: Absorption

8

spectrum of apo-gR (0.06 % DDM, 50 mM citrate buffer pH 5 and 300 mM NaCl). 2→6:

9

absorption spectra at different time intervals after the addition of all-trans retinal. (b)

10

Difference spectra; the absorption spectrum obtained after 10 sec is subtracted from each

11

spectrum. (c) Kinetic traces and monitored absorption at 585 nm; the solid line represents

12

the fitted line.

13

Figure 7. (a) The incubation process of retinal analogue 9 with apo-gR in the presence of

14

sal. 1: Absorption spectrum of apo-gR (0.06 % DDM; 50 mM citrate buffer pH 5 and 300

15

mM NaCl). 2: Absorption spectrum after the addition of sal. 3→6: Absorption spectra at

16

different time intervals after the addition of all-trans retinal. (b) Difference spectra; the

17

absorption spectrum obtained after 10 sec is subtracted from each spectrum. (c) The rate

18

of the process was detected by an absorption at 594 nm; the solid line represents the fitted

19

line.

20

Figure 8. (a) Homology modeling (side view) of the ribbon structure of the gR protein .

21

The retinal chromophore is shown as a stick model (green) (b) Top view of gR; the

22

retinal is attached to Lys-257 (c) Stick model of retinal in gR; the chromophore, shown


Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

from the cyclohexane ring side (front), is surrounded by aromatic residues within the van

2

der Waals distance. (d) Protein residues surrounding the protonated Schiff base of retinal

3

in gR.

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18



(a) 368

1

CHO

2

3

Absorbance

0.15

0.10

5

0.05

1

Apo 1 10 sec 4 min 32 min 16.5 hr 5

554

4 0.00

400

5

500

600

700

Wavelength(nm)

(b) 6

8

0.05

∆ Absorbance

7

1 10 sec

4

4

555

4 min 32 min 16.5 hr

1

0.00

9

10

388

-0.05

400

500

600

700

Wavelength(nm) 11

12

13

14

(c)

Norm. Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 38

0.9

0.6

0.3

15 0

20000

40000

60000

Time (sec) 16

17

Figure 1


Page 31 of 38

All trans retinal All trans retinal+sal

0.09

O.D.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.06

0.03 3

4

5

6

7

8

9

pH 1

2

Figure 2

3

4

5

6

7

8

9

10

11



1

(a) CHO

0.50

3

4

Absorbance

2

485

367

518

Apo 1 Apo+sal 10 sec 4 min 32 min 16.5 hr 6

6

0.25

1 5

0.00

400

500

600

700

Wavelength(nm) 6

(b)

1 10 sec

8

9

∆ Absorbance

7

0.06

4

15 16 17 18

400

565

4

500

600

700

Wavelength(nm)

(c) 1.0

12

14

486

390

-0.06

13

4 min 32 min 16.5 hr

0.00

10

11

518

1

Norm. Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 38

0.9 0.8 0.7

0

10000

20000

30000

Time (Sec) 19 20

Figure 3 Page 32 of 38 ACS Paragon Plus Environment

Page 33 of 38

(a) 368

3 4

Absorbance

2

CHO

0.3

1

0.2

Apo 1 10 sec 32 min 4.5 hr 16.5 hr 5

5

1

0.1

520

5

0.0

6

400

500

600

700

Wavelength(nm) (b)

8

0.10

9 10 11

12

∆ Absorbance

7

1

0.05

4

520

10 sec 32 min 4.5 hr 16.5 hr

4

1 0.00

-0.05

412

13

400

500

600

700

Wavelength(nm) 14 15 16 17 18

(c)

Norm. Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.9

0.6

0.3

19 20

0

30000

60000

90000

Time (sec) 21 22

Figure 4

23



1

(a) CHO

484

2

4 5

Absorbance

3

515

368

0.4

0.2

400

7

12 13

500

600

700

(b) 484

1 10 sec 52 min 3.9 hr 16.5 hr

0.10 ∆ Absorbance

11

6

Wavelength(nm)

9 10

1

1 0.0

8

Apo Apo+sal 10 sec 52 min 3.9 hr 16.5 hr

6

6

0.05

518

4 555

4

1

0.00

-0.05

392

14

400

500

600

700

Wavelength(nm)

15

(c) 16

17

18

19

20

Norm. Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

0.96

0.84

0.72 0

50000

100000

150000

Time (sec) 21

Figure 5


Page 35 of 38

(a) 1

3

CHO N

Absorbance

2

355

0.3

1

457

Apo 10 sec 32 min 52 min 1.9 hr 13.5 hr 6

0.2 6 1

0.1

4

0.0

5

400

500

600

700

Wavelength(nm) 6

(b)

10 11

∆ Absorbance

8 9

585

0.06 1 10 sec

7

0.03

5

5

32 min 52 min 1.9 hr 13.5 hr

1

0.00

-0.03

338 460

12

400

500

600

700

Wavelength(nm)

13

(c) 14 15 16 17 18

Norm. Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.9

0.6

0.3

19 0

15000

30000

20

Time (sec)

21

Figure 6

45000



1

(a) CHO

2

3 4

Absorbance

0.6

486

N

362

0.4 6

Apo 1 Apo+sal 10 sec 32 min 1.5 hr 16.5 hr 6

0.2 1

5 0.0

400

6

500

600

700

Wavelength(nm)

(b) 1

7

9

10

∆ Absorbance

8

594

10 sec 32 min 1.5 hr 4 16.5 hr

0.06

523

4 1

0.03

0.00 440

-0.03 11

400

500

600

700

Wavelength(nm) 12

(c) 0.9

13

14

15

Norm. Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

0.6

0.3

16

0

15000

30000

45000

Time (sec) 17

Figure 7


Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 8

3

4

5

6

7

8



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 38 of 38

Table of Contents (TOC) Image

2

3


Retinal Binding to Apo-Gloeobacter Rhodopsin. The Role of pH and

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