Unique Hydrogen Bonds in Membrane Protein Monitored by Whole

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Unique Hydrogen Bonds in Membrane Protein Monitored by Whole Mid-IR ATR Spectroscopy in Aqueous Solution Shota Ito, Masayo Iwaki, Shinya Sugita, Rei Abe-Yoshizumi, Tatsuya Iwata, Keiichi Inoue, and Hideki Kandori J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11064 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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The Journal of Physical Chemistry

Unique Hydrogen Bonds in Membrane Protein Monitored by Whole Mid-IR ATR Spectroscopy in Aqueous Solution Shota Ito1, Masayo Iwaki1, Shinya Sugita1, Rei Abe-Yoshizumi1, Tatsuya Iwata1, Keiichi Inoue 1, 2, 3,

1.

Hideki Kandori* 1, 2

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-

ku, Nagoya 466-8555, Japan, 2.

OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya

466-8555, Japan, 3.

PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-

0012, Japan

Corresponding Author * [email protected]

ACS Paragon Plus Environment

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The Journal of Physical Chemistry 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

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ABSTRACT

Protein function is coupled to its structural changes, for which stimulus-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful method.

By optimizing the

attenuated tonal reflection (ATR)-FTIR analysis on the sodium pumping rhodopsin KR2 in aqueous solution, we first measured the accurate difference spectra upon sodium binding in the whole IR region (4000-1000 cm-1). The new spectral window allows the analysis of not only the finger print region (1800-1000 cm-1), but also the hydrogen-bonding donor region (4000-1800 cm-1), revealing an unusually strong hydrogen bond of Tyr located in the sodium binding site of KR2.

Progress in ATR-FTIR difference spectroscopy provides stimulus-induced structural

changes of membrane proteins under physiological aqueous conditions.


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1


Introduction

2

Chemical reactions inside a protein are usually controlled by the alteration of a hydrogen

3

bonding network composed of amino acids and internal water molecules. Understanding how

4

chemical reactions are coupled with protein structural changes has great demand in the life

5

sciences. High resolution X-ray crystallography and cryo-EM tomography can detect the 3D

6

structure of proteins, including water molecules.1,

7

simulation led to a deeper understanding of protein dynamics at an atomic resolution.3,

8

However, it is always in question whether structural dynamics in a crystal can be monitored

9

under physiological conditions. In this sense, NMR may be the only experimental method to

10

probe the hydrogen-bonding alteration of biomolecules in aqueous solution, though its

11

application to membrane proteins has been limited.5

2

Progress of free electron laser and MD 4

12

Vibrational spectroscopy is not regarded as a tool in structural biology, as it cannot determine

13

atomic positions in biomolecules. Nevertheless, stimulus-induced difference Fourier-transform

14

infrared (FTIR) spectroscopy is a powerful method to investigate protein structural changes

15

accompanying a chemical reaction. This method has been widely applied to various photo-

16

reactive proteins, where the stimulus is light.6-8 Progress in light-induced difference FTIR

17

spectroscopy has allowed the frequency region to be extended from the conventional fingerprint

18

region (1800-800 cm-1) into the whole mid-IR region (4000-800 cm-1), and can monitor

19

hydrogen-bonding donors such as O-H and N-H stretches.9,

20

stretching vibration is gigantic, but such an extension was achieved by reducing the water

21

content in a sample without affecting function, such as hydrated films. This provides useful

22

information about hydrogen-bonding alteration during functional processes in rhodopsins, flavo-

23

protein and photosystem II.9-12 In fact, stretching vibrations of protein bound water molecules in

10

Absorption of water O-H


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Page 4 of 21

1

animal and microbial rhodopsins were monitored in the whole mid-IR region, exposing a unique

2

role of protein-bound water molecules in rhodopsins.9, 10, 13-15

3

While light-induced difference FTIR spectroscopy was successfully used to study the

4

structure/function of photoreceptive proteins, its extension to general proteins has not been easy,

5

because light is an ideal stimulus. For other stimuli, attenuated total reflection (ATR)-FTIR

6

spectroscopy can be used. ATR-FTIR is able to measure difference spectra by exchanging

7

solutions, leading to ion- or ligand-induced structural changes in an aqueous solution.16-19

8

However, strong water absorption of a bulk solution prevents analyses in the whole mid-IR

9

region (4000-1000 cm-1). It was possible to measure X-H stretch in D2O,20 but only a few

10

reports monitored the O-H stretch at >3550 cm-1 in H2O,21, 22 the frequency region of weakly

11

hydrogen-bonded (or dangling bond) water molecules. Accurate measurements for the entire

12

mid-IR region are a real challenge in FTIR spectroscopy of biomolecules.

13 14

Materials and Methods

15

The KR2 gene, whose codons were optimized for E. coli expression, was synthesized. KR2

16

WT, isotope-labeled and mutant proteins with six histidines at the C-terminus, were expressed in

17

E. coli C41 (DE3) strain on M9 media. Although we did not measure isotope incorporation by

18

mass spectrometry, the present vibrational data show high yield of incorporation (see Fig. 3

19

below). The protein was purified by a Co-affinity column (TALON, Qiagen) chromatography as

20

described previously and solubilized in 0.1% n-dodecyl-β-D-maltoside (DDM).23 KR2 was

21

reconstituted into a mixture of POPE and POPG (3:1) (molecular ratio of KR2:lipid = 1:20). The


4

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KR2 sample reconstituted into POPE/POPG liposomes was washed three times with 2 mM Tris

2

buffer containing 1 mM NaCl (pH 8.5).

3

The proteins reconstituted into lipids were placed on the surface of a silicon ATR prism

4

(Smiths, three effective internal reflections) and naturally dried. The sample was then rehydrated

5

with the first solvent, containing 100 mM NaCl in 20 mM Tris-H3PO4 (pH 8.0), at a flow rate of

6

0.5 ml/min. As we reported the Kd value to be 11.4 mM for KR2,23 we used 100 mM NaCl in

7

this study. The ATR-FTIR spectra (an average of 352 interferograms) was recorded at 293 K

8

and a 2 cm-1 resolution with a Bio-Rad FTS-7000 spectrometer, equipped with a liquid nitrogen-

9

cooled MCT detector. After exchange to the second solvent (100 mM CsCl in 20 mM Tris-

10

H3PO4 at pH 8.0), the ATR-FTIR spectra were recorded. In the first paper, the NaCl minus RbCl,

11

and NaCl minus CsCl spectra were identical, whereas NaCl minus KCl spectra were slightly

12

different,23 suggesting that K+ may bind to KR2 partially, unlike Rb+ and Cs+. Therefore, we

13

used Cs+, not K+, as the control of the unbound form. In the experiments, the cycling procedure

14

was repeated 8-10 times, and the difference FTIR spectra were calculated between the first and

15

second solvents. We also measured NaCl minus CsCl spectra without the KR2 sample, and

16

effect of water absorption between Na+ and Cs+ was corrected.

17 18

Results and Discussion

19

Here, we report precise difference FTIR spectra in the whole mid-IR region upon sodium-

20

binding to Krokinobacter rhodopsin 2 (KR2), a light-driven sodium pump rhodopsin.23 In our

21

previous study, we detected spectral changes of the water O-H stretch at 3630-3610 cm-1 in an

22

aqueous solution upon chloride-binding to pharaonis halorhodopsin (pHR), a light-driven


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chloride pump rhodopsin.22 In the measurements, we used a 9-reflection diamond cell, which

2

provides high absorbance. This is important to obtain difference spectra with a good S/N ratio.

3

We were able to obtain precise difference spectra in the 1800-1000 cm-1 region, while strong

4

water absorption at 3500-3200 cm-1 hampered the ability to obtain precise difference spectra

5

(Fig. S1). In contrast, use of the 3-reflection silicon cell reduced absolute absorption in this

6

study (Fig. 1a), so that difference spectra between 100 mM NaCl and CsCl (solid line in Fig. 1b),

7

but not between 100 mM NaCl minus NaCl (dotted line in Fig. 1b), were precisely obtained in

8

the whole mid-IR region (detailed spectra at 4000-2000 cm-1 in Fig. S2). All vibrational bands in

9

Fig. 1b originate from the difference between sodium-bound and sodium-unbound states of KR2.

10 11 12 13 14 15 16 17 18 19 20

Figure 1. (a) ATR-FTIR spectra of KR2 in 100 mM NaCl 20 mM Tris-H3PO4 pH 8 at 293 K in

21

the whole middle infrared region (4000-1000 cm-1). One division of the y-axis corresponds to

22

0.5 absorbance units. (b) Ion binding-induced difference infrared spectra of KR2 in 100 mM

23

NaCl minus CsCl (solid line), 100 mM NaCl minus NaCl (dotted line) with 20 mM Tris-H3PO4

24

pH 8 buffer. One division of the y-axis corresponds to 0.001 absorbance units.


6

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1 2

Difference ATR-FTIR spectra of KR2 were reported in the 1800-1000 cm-1 region, which

3

clearly demonstrated a sodium binding signal on the extracellular side.23,

24

4

crystallography then determined the sodium binding site in KR2, being located at the boundary

5

of two molecules in the pentamer (Fig. 2).25 According to the structure, Y25, T83, F86, T87 and

6

D102 constitute the sodium binding site, which is consistent with the no sodium binding ATR-

7

FTIR signal for D102N.24 The hydrogen bonding network in the binding site must be involved

8

in the frequency region at 4000-2000 cm-1 (Fig. S2), and in fact this region provides useful

9

structural information.

X-ray

10 11 12 13 14 15 16 17 18 19

Figure 2. X-ray structure of the KR2 pentamer (PDB: 4XTN) and a close-up of the sodium

20

binding site on the extracellular side. Sodium ion (purple sphere) is surrounded by Y25, T83,

21

F86, and T87 of one monomer, and by D102 of another monomer and two water molecules

22

(green spheres).

23


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Characteristic vibrational bands of the ion binding-induced difference spectra in Fig. 1b were

2

assigned by use isotope-labeled proteins. Fig. 3 shows the difference spectra in the conventional

3

1800-1000 cm-1 region, where the unlabeled spectra (black curve) are compared with those of

4

15

5

reproduced from those reported previously.23 It should be noted that light-induced difference

6

spectra of all rhodopsins exhibit large spectral changes at 1200-1100 cm-1 owing to C-C

7

stretching vibrations of the retinal chromophore. Such spectral feature at 1200-1100 cm-1 is

8

much smaller for the ion binding-induced difference spectra (black curve in Fig. 3), being

9

consistent with the sodium binding site distant from the chromophore. In Fig. 3a, large spectral

N-labeled (a),

13

C-labeled (b), and Tyr-D4-labeled (c) proteins. The unlabeled spectra are

10

difference was seen at 1550-1500 cm-1, characteristic frequency of amide-II vibration.

11

contrast, amide-I vibration at 1700-1600 cm-1 is much less sensitive to the 15N-labeling, which is

12

consistent with the spectral feature in Fig. 3a. In addition, isotope shift was observed for the

13

bands at 1692(+)/ 1675(-) cm-1, and at 1127(+)/ 1116(-)/ 1094 (-)/ 1079(+) cm-1, which are

14

assignable for the vibrations of side chains of arginine and histidine, respectively.26-29

15

Most of bands show isotope shifts upon

13

In

C-labeling in Fig. 3b, and no shifts originate from

16

either (i) vibrations that do not contain motion of carbon atom, or (ii) vibrations of the

17

chromophore retinal and phospholipids. When we reported the NaCl-minus-KCl difference

18

ATR-FTIR spectra, we observed a peak pair at 1748(+)/ 1727(-) cm-1,23 characteristic frequency

19

of protonated carboxylic acids. Similar bands were observed at 1749(+)/ 1732(-) cm-1 in this

20

study, whereas there are no isotope shift of upon 13C-labeling, implying that these vibrations do

21

not originate from protonated carboxylic acids. The bands at 1749(+)/ 1732(-) cm-1 are probably

22

ascribable for C=O stretch of lipids, and the present study demonstrates that the sodium-binding

23

to KR2 accompanies structural perturbation of lipid bilayers. The bands at 1250(+)/ 1232(-) cm-1


8

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1

in Fig. 3b are located at characteristic frequency of C-O stretch of tyrosine,26,

30

2

confirmed by the measurement of Tyr-D4-labeled proteins. Fig. 3c shows that half of the

3

positive 1250-cm-1 band originates from the vibration of tyrosine. In addition, the positive band

4

at 1511 cm-1 also contains vibration of tyrosine, as it shifts to 1429 cm-1 upon Tyr-D4-labeling.

5

From the literature, the phenolic O-H group acts as both hydrogen-bonding donor and acceptor.31

which is

6 7 8 9 10 11 12 13

Figure 3. Ion binding-induced difference infrared spectra of KR2 in the 1800-1000 cm-1 region.

14

The difference spectra were recorded between 100 mM NaCl and CsCl with 20 mM Tris-H3PO4

15

pH 8 buffer. The black-colored spectra represent difference spectra of unlabeled KR2 protein,

16

whereas red-colored spectra represent difference spectra of KR2 15N (a), 13C (b), and Tyr-D4 (c)

17

labeled protein. One division of the y-axis corresponds to 0.005 absorbance units.

18


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O-H and N-H stretching vibrations under weak and moderate hydrogen bonds appear in the

2

frequency region in Fig. 4. All bands were shifted by 15N-isotope labeled protein, indicating that

3

all vibrations originated from N-H stretches. In other words, sodium-binding to KR2 does not

4

alter dangling bonded water signals, unlike NpHR and cytochrome c oxidase.21, 22 Protein-bound

5

water molecules are located in the sodium binding site (Fig. 2),25 while the present study

6

suggests that these water molecules do not form specific hydrogen bonds, but form hydrogen

7

bonds as in aqueous solution. The 3348(+)/ 3327(-) cm-1 and 3299(+)/ 3273(-) cm-1 bands are

8

assignable to N-H stretch of protein backbone (amide-A).32 The upshifted N-H stretch shows

9

that the internal hydrogen bond between α-helical pitches weakens upon sodium binding. The

10

bands at 3496(+), 3455(+) cm-1 were also shifted by

15

11

some N-H stretch weakens the hydrogen bond more than the unbound state. N-D stretching

12

frequencies of Arg82 in a light-driven proton-pump bacteriorhodopsin were reported to be 2579

13

and 2292 cm-1,33 whose N-H stretches correspond to ~3550 and ~3100 cm-1, respectively. Thus,

14

the bands at 3496(+), 3455(+) cm-1 may be ascribable for N-H stretch of Arg under weak

15

hydrogen-bonding conditions. On the other hand, 13C- and Tyr-D4-labeled proteins did not affect

16

the X-H vibration in the 3540-3150 cm-1 region.

N labeled protein, which indicated that

17 18 19


10

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1 2 3 4 5 6 7 8 9


10


11

pH 8 buffer. The black spectra represent difference spectra of unlabeled KR2 protein, whereas

12

red spectra represent difference spectra of KR2

13

One division of the y-axis corresponds to 0.0015 absorbance units.

15

N (a),

13

C (b), and Tyr-D4(c) labeled protein.

14 15

Although Fig. 1a shows almost no absorption at 2800-2400 cm-1, O-H and N-H stretching

16

vibrations of tyrosine and histidine under strong hydrogen bonds have been reported for sensory

17

rhodopsin I, photosystem II, and the BLUF domain.34-36 Similar spectral features were observed

18

for KR2. Fig. 5 shows the appearance of five positive bands at 2805, 2765, 2696, 2629 and 2512

19

cm-1 upon sodium binding. The 2805 cm-1 band seems to have shifted upon

20

5a), while all bands shifted in response to

21

The results in Fig. 5c allow for the identification of these vibrations from the O-H stretch of Tyr.

15

N labeling (Fig.

13

C (Fig. 5b) and Tyr-D4 (Fig. 5c) labeled proteins.


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The phenolic O-H stretching vibration appears at ~3600 cm-1 and ~3200 cm-1 under no

2

hydrogen-bonding and strong intramolecular hydrogen-bonding conditions, respectively.37

3

Therefore, the observed frequencies are extremely low as the phenolic O-H stretch of Tyr.

4

Nevertheless, similar broad positive peaks with four peaks at 2800-2400 cm-1 were observed for

5

the BLUF domain, and were interpreted as the formation of an unusually strong hydrogen bond

6

of Tyr with Gln during the photoactivation intermediate.36 In the case of the BLUF domain,

7

multiple peaks were interpreted as Fermi resonance, where the O-H stretch is coupled with

8

overtone vibrations of the phenol ring. A very similar spectral feature strongly suggests the same

9

origin. Corresponding negative O-H stretch was unclear from the data (Figs. 4 and 5), being

10

possibly masked by the strong positive signals.

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1 2 3 4 5 6 7 8 9 10 11 12 13


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pH 8 buffer. The black spectra in (a)-(e) represent difference spectra of unlabeled KR2 protein,

16

whereas red spectra represent difference spectra of KR2

17

protein. Red spectra in (d) and (e) represent the D102N and Y25F mutants, respectively. One

18

division of the y-axis corresponds to 0.0004 absorbance units.

15

N (a),

13

C (b), and Tyr-D4(c) labeled

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From the structure of KR2, Y25 is a strong candidate for the observed signal of Tyr, and the

2

phenolic oxygen of Y25 is located at 2.5 Å from an oxygen of D102 (Fig. 2). The distance is

3

shorter than the normal oxygen-oxygen hydrogen bond, suggesting a strong interaction with a

4

negatively charged D102. A similar short distance was reported between Y42 and E46 (2.5 Å)

5

of photoactive yellow protein, whose hydrogen bond is unusually strong (low barrier hydrogen

6

bond).38 In KR2, a strong hydrogen bonding interaction between Y25 and D102 must play an

7

important role in sodium binding. In fact, Fig. 5d and 5e show that mutation of Y25 (Y25F) and

8

D102 (D102N) completely abolishes sodium binding to KR2 (see Fig. S3 for the 1800-1000 cm-1

9

region). It is noted that sodium binding is not necessary for the sodium pump function of KR2,23

10

whereas sodium binding largely contributes to increase the thermal stability of KR2.24 Thus, an

11

unusually strong hydrogen bond of Y25 with D102 must contribute to the protein’s stability.

12 13

Conclusion

14

In conclusion, we report the first ATR-FTIR difference spectra in the whole infrared spectral

15

region by ion-exchange methods. Expansion of the infrared window deepens our understanding

16

of the hydrogen bonding network in the metal coordinated structure in protein. A combination

17

between this method, isotope labeling and the use of mutant proteins elucidated that Y25 forms

18

an unusually strong hydrogen bond to D102 in a light-driven sodium pump KR2. On the other

19

hand, protein-bound water molecules do not form specific hydrogen bonds as can be seen for

20

chloride pumping rhodopsin pHR.22 ATR-FTIR different spectra in the whole mid-IR region

21

will be widely applied for various ion-transport and ligand binding proteins to analyze details of

22

the hydrogen bonding network around a substrate binding site in the future.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: ATR-FTIR spectra of KR2 recorded on 9-reflection diamond prism, ATR-FTIR different spectra upon sodium binding in the 4000-2000 cm-1 region, and ATR-FTIR different spectra of mutant proteins upon sodium binding in the 1800-1000 cm-1 region.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to K.I. (26708001, 26115706, 26620005) and to H.K. (25104009, 15H02391).


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Unique Hydrogen Bonds in Membrane Protein Monitored by Whole

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