Design of New Extraction Surfactants for Membrane Proteins from

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Design of New Extraction Surfactants for Membrane Proteins from Peptide-Gemini Surfactants (PG-surfactants) Masahide Shibata, Shuhei Koeda, Tomoyasu Noji, Keisuke Kawakami, Yuya Ido, Yuichi Amano, Naoki Umezawa, Tsunehiko Higuchi, Takehisa Dewa, Shigeru Itoh, Nobuo Kamiya, and Toshihisa Mizuno Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00417 • Publication Date (Web): 29 Aug 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Bioconjugate Chemistry

Design of new extraction surfactants for membrane proteins from peptide gemini surfactants (PG-surfactants).

Masahide Shibata †, Shuhei Koeda †, Tomoyasu Noji ‡, Keisuke Kawakami ‡, Yuya Ido †, Yuichi Amano §, Naoki Umezawa §, Tsunehiko Higuchi §, Takehisa Dewa †, Shigeru Itoh †, Nobuo Kamiya ‡, Toshihisa Mizuno †*

†

Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho

Showa-ku, Nagoya, Aichi 466-8555, Japan ‡

Osaka City University, The OCU Advanced Research Institute for Natural Science &

Technology (OCARINA), 3-3-138 Sugimoto-cho, Sumiyoshi, Osaka 558-8585, Japan §

Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1

Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan

*Corresponding Author E-mail Address: [email protected] (Toshihisa Mizuno) Tel & Fax: +81-52-735-5237

1 ACS Paragon Plus Environment


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Abstract The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK,

from

the

peptide-gemini-surfactant

(PG-surfactant)

molecular

framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues (Figure 1). Using Photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus (T.) vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins, while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native-PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins.


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NH

HN

O

O S

Y Cys

S X

Cys NH2

Figure 1. Chemical structure of PG-surfactants with C12 alkyl chains



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Introduction One-third of natural proteins are classified as membrane proteins, and these have crucial roles in various biological events occurring at membranes, e.g., cell adhesion,1 secretory protein trafficking,2 signal transduction via ligand recognition of GPCR,3 ATP synthesis,4 and photosynthesis.5 To understand the precise relationship between biological functions and protein structures, it is important to isolate target membrane proteins from biological membranes, while maintaining their original supramolecular structures and functions. Owing to recent advances in cryptographic structural analysis using X-ray free electron laser (XFEL),6 the importance of methods to enable the stable and efficient isolation of target membrane proteins from biological samples can be re-examined. Extraction surfactants are generally utilized to extract proteins from biological membranes and to separate these proteins from other membrane components.7 Extraction surfactants must have two functions, i.e., they must extract membrane proteins from biological membranes and solubilize the extracted membrane proteins in an aqueous buffer. Upon both processes, these surfactants should not destruct the native supramolecular structure of target membrane proteins. Most surfactants could break non-covalent interactions, such as hydrogen bonds, hydrophobic interactions, and salt-bridges, which are indispensable to maintain supramolecular protein structures of membrane proteins. Accordingly, the kinds of effective extraction surfactants are limited, and this may explain why membrane protein research has been impeded in comparison to research on water-soluble proteins.8 We recently developed a novel class of solubilization surfactants for membrane proteins based on a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified -modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of dodecylamidemethylene-modified Cys residues.9, 10

We named this scaffold the “peptide gemini surfactant” (PG-surfactant, Figure 1) in

our previous study.11 Choosing the betaine peptide sequence -Asp-Lys-Asp-Lys- as the linker peptide (-X-) and the N-terminal acetylated Asp or Lys as the peripheral peptide (Y-), we successfully constructed DKDKC12K and DKDKC12D as new solubilization surfactants for membrane proteins.10 Using photosystem I and II (PSI and PSII) from Thermosynecoccus elongatus12 and T. vulcanus13 as representative membrane proteins, we proved that these surfactants can solubilize PSI and PSII without affecting the supramolecular structures based on detail assessments of photophysical properties. 4 ACS Paragon Plus Environment

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However, probably owing to the rather hydrophilicity and flexibility of the linker peptides of DKDKC12K and DKDKC12D, these surfactants could not extract PSI and PSII from thylakoid membranes and accordingly are not qualified to be extraction surfactants for membrane proteins. In this study, in order to improve extraction efficiency, we examined another series of PG-surfactants with different linker peptide sequences. Specifically, we tested those forming a β-turn structure. A hairpin structure at the linker peptide of PG-surfactants could facilitate the parallel alignment of C12 alkyl chains; accordingly, PG-surfactants with a β-turn-forming peptide might have an improved ability to stick to and disrupt biological membranes, improving the membrane protein extraction efficiency. In natural proteins, β-turn structures are composed of three to five residues. Searle et al. reported that the -Asn-Pro-Asp-Gly- sequence forms a β-turn structure as an alternative to the G-bulged turn sequence -Thr-Leu-Thr-Gly-Lys- at the N-terminal side of natural ubiquitin.14-16 The non-natural peptides, containing dinipectoic acid, have a tendency to form a β-turn-like structure.17 In particular, those with the -βAla-Nip(-)-Nip(+)-βAlasequence form a stable β-turn-like structure, even in an aqueous solution.18 Therefore, we constructed a series of PG-surfactants with the above β-turn-forming peptides as a linker peptide and evaluated their abilities both to solubilize membrane proteins in an aqueous buffer and to extract membrane proteins from biological membranes.



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Figure 2. Chemical structures of ß-turn forming peptides


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Results and Discussion Design of extraction surfactants for membrane proteins from the PG-surfactant molecular framework PG-surfactants

are

amphiphilic

molecules

composed

of

two

long

alkyl-chain-modified Cys residues, a linker peptide (-X-), and a peripheral peptide (Y-) (Figure 1).11 In conventional gemini surfactants, linear alkanes or oligoethylene chains are usually adopted as a linker of two molecules of single-chain surfactants. Owing to dimerization of single-chain surfactants, critical aggregation concentrations (CACs) of gemini surfactants are generally smaller than those of the parent single-chain surfactants.19 As for PG-surfactants, short linear peptides function not in linking two long alkyl chains into a single molecule, but also as a hydrophilic head group.9-11 To endow PG-surfactants with amphiphilic properties, choice of hydrophilic linker peptides is indispensable. However, by adopting hydrophilic peptides at the peripheral peptide (Y-), less hydrophilic peptides can be chosen at a linker peptide, without diminishing solubility in an aqueous buffer, enabling a flexible design of various functional PG-surfactants. To extract membrane proteins from biological membranes, surface-active functions of surfactants are generally applied to disrupt lipid membrane integrity and separate membrane protein molecules from other membrane components.8 After detachment of membrane lipid molecules, these surfactants function to solubilize the extracted membranes proteins in an aqueous buffer by covering the exposed hydrophobic membrane integral domains with surfactant micelles. However, if common ionic surfactants, such as sodium dodecyl sulfate (SDS), were adopted for this process, protein denaturation owing to the disruption of non-covalent interactions, such as hydrophobic interactions, salt-bridges, and hydrogen bonds, essential to build-up unique protein supramolecular structures, would occur, which is not appropriate for the isolation of membrane proteins from biological membranes. Extraction surfactants are generally utilized to extract membrane proteins. Surfactants that include zwitterionic or nonionic hydrophilic groups, such as N-oxide and sugars, as a polar head group, exhibit better properties in extraction efficiency and maintenance of protein structures.7,

20

However, those including peptide sequences are rather limited.21 If we designed peptide-containing extraction surfactants, owing to the ease of choosing peptide sequences in surfactants, it may be possible to evolve many new extraction surfactants 7 ACS Paragon Plus Environment


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with different properties. Furthermore, additional interactions of peptide moieties in surfactants with amino acid residues in membrane proteins could influence protein stability and extraction specificity. Therefore, the development of peptide-containing extraction surfactants should be an attractive study target. Meanwhile, we recently successfully constructed a novel class of solubilization surfactants for membrane proteins, DKDKC12K and DKDKC12D, by screening several hydrophilic peptide sequences at a linker peptide (-X-) and a peripheral peptide (Y-).10 However, unlike general extraction surfactants for membrane proteins, the extraction efficiency of DKDKC12K and DKDKC12D from biological membrane samples, i.e. thylakoid membranes, was quite low. In order to improve extraction efficiency, we designed another set of PG-surfactants, having a structured linker peptide that forms a β-turn structure in this study (Figure 2). Searle et al.14 designed the Asn-Pro-Asp-Gly sequence from the original peptide sequence Thr-Leu-Thr-Gly-Lys to form a β-turn structure at the N-terminal side of ubiquitin and its secondary structure was assigned by NMR spectroscopy. βAla-Nip(-)-Nip(+)-βAla also forms a β-turn-like secondary structure based on NMR spectroscopy and X-ray crystallography.18 Both peptide sequences are short enough to use as a linker peptide in PG-surfactants and so we designed a new series of PG-surfactants using these as a β-turn-forming linker peptide. Low hydrophilicity of these β-turn-forming peptides perhaps significantly decreases solubility of the PG-surfactants in an aqueous buffer. Therefore hydrophilic sequences, such as the acetylated Lys, (Lys)2, Asp, or (Asp)2, were tested as a peripheral peptide. The PG-surfactants studied in this study are summarized in Table 1. As a reference of those having Asn-Pro-Asp-Gly as a linker peptide, those having Asp-Pro-Asp-Gly were also examined. Mutation in the β-turn-forming peptide can decrease tendency to form a β-turn structure, and we would evaluate relationship between secondary structure of linker peptide and extraction efficiency. These new set of PG-surfactants, including Asn-Pro-Asp-Gly, Asp-Pro-Asp-Gly, or βAla-Nip(-)-Nip(+)-βAla, were synthesized on a resin for Fmoc solid-phase peptide synthesis, as similar to our previous studies, and these were purified by a reversed-phase high-performance liquid chromatography. These surfactants were assigned using electrospray ionization high-resolution mass spectrometry.


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Table 1 Summary of CAC values (µM) and hydrodynamic diameters (d, nm) of the PG-surfactants in this study a CAC

d (nm)b

PG-surfactant

-X-

Y-

DKDKC12K c

-Asp-Lys-Asp-Lys-

Ac-Lys-

8.3

6

DKDKC12D c

-Asp-Lys-Asp-Lys-

Ac-Asp-

7.9

6

NPDGC12

-Asn-Pro-Asp-Gly-

Ac-

---

---

NPDGC12D

-Asn-Pro-Asp-Gly-

Ac-Asp-

---

---

NPDGC12DD

-Asn-Pro-Asp-Gly-

Ac-(Asp)2-

---

---

NPDGC12K

-Asn-Pro-Asp-Gly-

Ac-Lys-

---

---

NPDGC12KK

-Asn-Pro-Asp-Gly-

Ac-(Lys)2-

33.7

5

DPDGC12KK

-Asp-Pro-Asp-Gly-

Ac-(Lys)2-

35.3

5

AβNp(-)Np(+)AβC12

-βAla-Nip(-)-Nip(+)-βAla-

Ac-

---

---

AβNp(-)Np(+)AβC12D


Ac-Asp-

25.3

6

AβNp(-)Np(+)AβC12K


Ac-Lys-

8.9

6

(µM)

a

In 50 mM phosphate buffer (pH 7) at 25 °C.

b

Hydrodynamic diameters were observed using 0.1 wt% PG-surfactant in 50 mM

phosphate buffer (pH 7) at 25 °C. c

These data are from reference.10



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Characterization of micelle formation properties of the set of PG-surfactants Prior to characterization of extraction efficiency, we first checked water solubility of the newly designed PG-surfactants in a neutral buffer, e.g., phosphate and Tris-HCl at pH 7. As similar to our previous study of DKDKC12,10 NPGDC12 lacking a hydrophilic peripheral peptide was insoluble in a neutral buffer, but NPDGC12KK, which had a di-cationic peptide Ac-(Lys)2- as a peripheral peptide showed sufficient solubility (>1 wt%). However, NPDGC12K, with mono-cationic Ac-Lys- did not exhibit enough solubility for practical use (1

wt%)

in

a

neutral buffer.

But

β

A Np Np A C12D having Ac-Asp- was less soluble in a neutral buffer. Micelle formation behaviors are studied for the water-soluble PG-surfactants. We performed dynamic light scattering measurements upon various concentrations from 1 to 0.01wt% in 50 mM phosphate buffer (pH 7) to characterize assembling morphologies in a neutral buffer. NPDGC12KK formed ~6–7-nm-diameter assemblies with a single fraction in the concentration range of 1 to 0.01 wt%, suggesting exclusive formation of micelle-like assemblies (Figure 3). Similarly, DPDGC12KK and AβNp(-)Np(+)AβC12K also formed only micelles with diameters of 6–7 nm (Figure 3) at the concentration range of 0.01–1 wt%. Since these surfactants only formed a micelle-like assembly, we determined their CAC values using the ANS fluorescence method. As shown in Figure 4, all surfactants showed a single inflection point at 5–30 µM, suggesting that each concentration of the infection points corresponds to each CAC. The calculated CAC values are summarized in Table 1. Some peptide-containing surfactants form various molecular assemblies with different morphologies, such as fibers, sheets, and bilayers.9, 22

However, the series of PG-surfactants in this study only form micelle-like assemblies.

This property is suitable to utilize as an extraction surfactant for membrane proteins since insertion into bilayer structure of biological membranes, destruction of their 10 ACS Paragon Plus Environment

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structural integrity, and solubilization of lipids and other components in a buffer are necessary to extract membrane proteins.

(b)

Volume

Volume

(a)

1

10

100 d (nm)

1

1000

10

100 d (nm)

1000

(c) 35 30 25 Volume (%)

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


20 15 10 5 0 1

10 d (nm)

100

1000

Figure 3. DLS profiles of the molecular assemblies of NPDGC12KK (a), DPDGC12KK (b), and AβNp(-)Np(+)AβC12K (c) on 0.01 wt% (black line), 0.1 wt% (red line), and 1 wt% (blue line) in 20 mM phosphate buffer (pH 7).



(b)

F

480

480

(a)

F

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-8

-7

-6

-5

-4

log [NPDGC12KK]

-3

-8

-7

-6

-5

-4

-3

log [DPDGC12KK]

(c)

Figure 4. Fluorescence intensity at 480nm of ANS (10 µM) in 50 mM phosphate buffer (pH 7) containing different concentrations (100 nM to 100 µM) of NPDGC12KK (a), DPDGC12KK (b), and AβNp(-)Np(+)AβC12K (c). The linear-fitting lines before and after the inflection point are also illustrated.


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Solubilization ability of membrane proteins with maintainance of protein structure and functions Prior to the membrane protein extraction experiments, we evaluated the solubilization behaviors of the new PG-surfactants for membrane proteins, since capability to solubilize membrane proteins with maintainance of native supramolecular structure and functions is the first requirement to be qualified as an extraction surfactant. As representative membrane proteins, we used PSI and PSII derived from T. vulcanus. PSI of T. vulcanus12 is a supramolecular pigment-protein complex consisting of 12 protein subunits, 96 Chl a molecules, 3 [4Fe-4S] clusters, etc. In a thylakoid membrane, it forms a trimer. PSII of T. vulcanus13 is a dimeric supramolecular complex composed of 20 protein subunits, 35 Chl a molecules, 1 Mn4CaO5 cluster, etc. In a thylakoid membrane, it forms a dimer. Because PSI and PSII contain many chromophores, such as Chl a, pheophytin, and carotenoids, and characteristic metal complexes, such as [4Fe-4S] clusters and oxygen evolution complex Mn4CaO5, in their supramolecular protein frameworks, a coincidence of photophysical properties is strong proof that PSI and PSII maintain their original protein structures. Furthermore, by examining photophysical properties, we can determine the areas of membrane proteins (membrane integral domain or extracellular domain) that are damaged. This easy-to-assess property is different from other membrane proteins, such as receptors, ion channels, and ion pumps, and it could be used to supply fruitful information to assess new extraction surfactants. To assess the effects of each PG-surfactant on protein structure, the surfactant-exchange method was applied as following the methods in our previous study.10 Purified PSI or PSII solubilized in a buffer [buffer D (20 mM HEPES-NaOH (pH 7.0), 10 mM MgCl2, 25% (w/v) glycerol) for PSI and buffer B (20 mM MES-NaOH (pH 6.0), 20 mM NaCl, 3 mM CaCl2, 25% (w/v) glycerol) for PSII] containing 0.1 wt% β-DDM was once precipitated using PEG1450. After washing off β-DDM by several washes in a buffer the resulting PSI or PSII precipitate was resolubilized in the same buffer containing 0.1 wt% of each PG-surfactant. Figures 5 and 6 summarize the absorption spectra of PSI and PSII solubilized in a buffer containing 0.1 wt% β-DDM or PG-surfactants, such as NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K. In any cases, the absorption spectra of PSI and PSII were all consistent with those using 0.1wt% β-DDM. Since PSI and PSII is known able to be 13 ACS Paragon Plus Environment


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solubilized in a buffer containing 0.1wt% β-DDM with a native state, it indicated that PSI and PSII could be solubilized without protein denaturation by NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K, respectively. The observed absorption spectra were mainly originated from the Chl as and carotenoids, buried in the membrane integration domains of PSI and PSII. So these consistencies implied maintainance of native state at the membrane integral domain of PSI and PSII. In case of PSI, the fluorescence spectrum at 77 K of PSI is also useful to analyze the degree of denaturation in the antenna Chl a conformations at the membrane integral domain.23 Therefore, we next observed fluorescence spectra of PSI at 77 K upon using the new series of PG-surfactants. The results are summarized in Figure 7. If protein denaturation was not occurred for the antenna Chl a conformations at the membrane integral domain, a fluorescence band was solely observed at 710 nm derived from the red-Chl a of PSI. But if damaged, another spectral band at 680 nm, corresponding to the eliminated free Chl a, could be observed. However, none of the fluorescence spectra having a spectral peak at 680 nm was observed upon using any new series of PG-surfactants NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K, which also suggests that these surfactants could solubilize PSI with keeping a native-state at the membrane-integral domain. To examine the degrees of denaturation at the extracellular domains of PSI and PSII, such as the [4Fe-4S] clusters Fa and Fb in PsaE for PSI and the oxygen-evolution complex (OEC) for PSII, we examined the light-induced electron transfer activities of PSI and PSII and the lifetimes of the charge-separated state at the special pairs P700 of PSI. The light-induced electron transfer activities of PSI and PSII can be estimated by the rates of O2 consumption and O2 evolution using an O2 electrode, respectively.10, 24-26 The lifetime of P700+• can be estimated from a non-linear curve-fitting analysis of the time-course decay in A689 after a pulse of photo excitation at 400 nm.10 The elimination or damage to PsaE, including the [4Fe-4S] clusters Fa and Fb shortens the lifetime of P700+• to 1 ms, instead of 30 ms (on native-state). We also used the data of PSI and PSII upon solubilization with 0.1 wt% β-DDM as a control of native state. The results are

summarized

in

Table

2.

Using

NPDGC12KK,

DPDGC12KK,

and

AβNp(-)Np(+)AβC12K, the light-induced electron transfer activities of PSI were observed by 44, 49, and 41 PSI-1 s-1, respectively, which are similar to that observed using


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β-DDM (41 ± 2.1e- PSI-1 s-1). Furthermore, the 1-ms fractions of the P700+• lifetime, corresponding to denaturation at the extracellular domain of PSI, were not observed using these surfactants. These data strongly suggested that NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K can solubilize PSI with a native-state at the extracellular domain as well as at membrane integral domain. Using NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K to solubilize PSII, the photo-induced electron transfer activities of PSII were estimated as 152, 160, and 140 PSII-1 s-1, which were also similar to that observed using β-DDM (156 ± 0.9 e- PSII-1 s-1). This indicates that NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K can also solubilize PSII without denaturation, not only at integral membrane domains, but also at extracellular domains.



(a)

(b) 1.6 Absorbance (a.u.)

Absorbance (a.u.)

1.6 1.2 0.8 0.4

1.2 0.8 0.4 0

0 400

500 600 700 Wavelength (nm)

400


800

400


800

(c) 1.6 Absorbance (a.u.)

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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1.2 0.8 0.4 0 800

Figure 5. Comparison of absorption spectra of PSI after solubilization in a buffer containing 0.1wt% β-DDM (black line) and NPDGC12KK (a), DPDGC12KK (b), or AßNp(-)Np(+)AßC12K (c) (red line); [PSI] = 24 nM, 40 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2 containing 0.1wt % surfactant at ambient temperature.


Page 17 of 35

(a)

(b)

1.6 Absorbance (a.u.)

Absorbance (a.u.)

1.6 1.2 0.8 0.4 0 400

500

600

700

800

Wavelength (nm)

1.2 0.8 0.4 0 400


800

(c) 1.6 Absorbance (a.u.)

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 0.8 0.4 0 400


800

Figure 6. Comparison of absorption spectra of PSII after solubilization in a buffer containing 0.1 wt% β-DDM (black line) and NPDGC12KK (a), DPDGC12KK (b), or AβNp(-)Np(+)AβC12K (c) (red line); [PSII] = 32 nM, 40 mM MES (pH 6.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1wt % surfactant at ambient temperature.


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Fluorescence Intensity (a.u.)


650


850

Figure 7. Fluorescence spectra of PSI at 77 K upon solubilization in a buffer containing 0.1 wt% β-DDM (black line), NPDGC12KK (red line), DPDGC12KK (blue line), and AßNp(-)Np(+)AßC12K (green line); [PSI] = 24 nM, 40 mM HEPES (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, and 0.1wt % surfactant.


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Table 2. Light-induced electron transfer activities of PSI and PSII and P700+• lifetimes of PSI upon solubilization with PG-surfactants. PG-surfactant

Light-induced electron transfer activity of PSI (e- PSI-1 s-1) a

Light-induced electron transfer activity of PSII (e- PSII-1 s-1) b

Lifetime of P700+• (ms) (ratio of each lifetime fraction, % )

β-DDM c

41 ± 2.1

156 ± 0.9

1ms (0%), 30 ms (60%), 150 ms (40%)

DKDKC12K c

42 ± 0.7

153 ± 3.6

1ms (0%), 30 ms (37%), 36 ms (63%)

DKDKC12D c

44 ± 1.5

144 ± 4.9

1ms (0%), 30 ms (49%), 50 ms (51%)

NPDGC12KK

44 ± 1.4

152 ± 7.0

1ms (0%), 30 ms (48%), 392 ms (52%)

DPDGC12KK

49 ± 1.7

160 ± 0.3

1ms (0%), 30 ms (18%), 289 ms (82%)


41 ± 0.7

140 ± 9.1

1ms (0%), 30 ms (62%), 101 ms (38%)

a

The concentration of PSI solubilized with 0.001wt% surfactant was 24 nM. A buffer containing 40 mM HEPES−NaOH (pH 7.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, and 0.4 M sucrose supplemented with 0.5 mM dichloroindophenol, 2 mM sodium ascorbate, and 0.5 mM MV2+ was used for oxygen uptake measurements. b The concentration of PSII solubilized with 0.001wt% surfactant was 24 nM. A buffer containing 40 mM MES−NaOH (pH 6.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, 0.4 M sucrose, and 0.5 mM phenyl-p-benzoquinone was used for oxygen evolution measurements. c The time-course flash-induced A698 changes, initiated by a xenon lamp flash (300 W, 5-µs pulse), were measured to estimate the decay rates of P700+•. [PSI] = 24 nM, 40 mM HEPES buffer (pH 7.5), 100 mM NaCl, 15 mM MgCl2, 15 mM CaCl2, containing 0.1wt% of surfactant.

19

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Evaluation as an extraction surfactant for membrane proteins from the extraction experiment of PSI and PSII from thylakoid membranes In the last section, we proved that the PG-surfactants containing β-turn-forming peptides NPDGC12KK, DPDGC12KK, and AβNp(-)Np(+)AβC12K could solubilize PSI and PSII without protein denaturation, as similar to the previously designed PG-surfactants, DKDKC12K and DKDKC12D. Therefore, we next performed extraction experiments of PSI and PSII from thylakoid membranes of T. vulcanus. Following the protocol by Shen et al.32 we used thylakoid membrane samples after removing most of membrane-bound water-soluble proteins derived from phycobilisome by several washes in buffer C. So, the thylakoid membrane samples used in this experiment contained mainly PSI and PSII molecules embedded in membranes, but residual membrane-bound proteins derived from phycobilisome were included. Using 1 wt% of PG-surfactants in buffer D, an extraction experiment was performed at 4 ˚C for 30-min with a gentle stirring. The colors of the extracted supernatants after centrifugation and the absorption spectra of these supernatants are summarized in Figure 8(a) and Figure 8(b), respectively. Based on previous studies, β-DDM is an effective surfactant to extract PSI and PSII from thylakoid membranes and the extraction yield using 1 wt% β-DDM was estimated by 32% based on a comparison of the Chl a contents between thylakoid membranes and the extracted fractions (Figure 8(b)). Probably because of higher hydrophilicity and flexibility of the linker peptides, DKDKC12K and DKDKC12D showed poor extraction ability (~0%) for Chl a-containing fractions, such as PSI and PSII. In contrast, NPDGC12KK showed a significant improvement in extraction efficiency reaching 28%, albeit it was a little less than that using β-DDM (31 %). Because DPDGC12KK with a weaker β-turn-forming peptide showed a substantial decrease in extraction efficiency (~2.4%), the choice of Asn-Pro-Asp-Gly sequence with a better β-turn forming tendency would function to improve extraction efficiency. However, AβNp(-)Np(+)AβC12K, with a different β-turn-forming peptide, did not show any improvement in extraction efficiency (~0%, i.e., it was as low as those of DKDKC12K and DKDKC12D). In case to use of AβNp(-)Np(+)AβC12K, several carotenoids from thylakoid membranes were extracted without leaving PSI and PSII in thylakoid membranes. These data implied that simple choice of a β-turn-forming peptide sequence to PG-surfactants does not ensure to have a better extraction


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efficiency. The proteins, extracted by NPDGC12KK and DPDGC12KK, were subjected to a hrCN-PAGE analysis and the results are illustrated in Figure 8(c). If the trimeric supramolecular structure of PSI and dimeric supramolecular structure of PSII were maintained during the extraction step, the expected molecular masses are 1016 kDa and 756 kDa, respectively.27 Using the extraction profile upon usage of 1 wt% β-DDM as a reference, each protein fraction band extracted by NPDGC12KK and DPDGC12KK was assigned as illustrated in Figure 8(c). The protein bands corresponding to the monomeric forms of PSI and PSII was observed for the extracted samples even the case to use 1wt% β-DDM in Blue native PAGE analyses. But it is known not mainly due to the destruction of trimeric or dimeric form of PSI or PSII but due to the concomitance of monomeric states of PSI and PSII stably in thylakoid membranes.28, 29 Therefore, the contamination of monomeric PSI and PSII in the extracted protein fractions does not mean that extraction surfactants cause serious damage to the supramolecular structures of PSI and PSII. By using 1wt% β-DDM as a control, we evaluated ratios between the extracted proteins, such as trimeric PSI, dimeric PSII, monomeric PSI and PSII, and other residual proteins from phycobilisomes. As results, NPDGC12KK dose not promote destruction of supramolecular structure of PSI and PSII, as similar to β-DDM (Table 3). DPDGC12KK, having fewer β-turn-forming peptide, seems able to extract trimeric PSI and dimeric PSII, selectively. However, owing to lower extraction ability residual proteins from the membrane-bound phycobilisome was preferentially extracted. These results indicated that the Asn-Pro-Asp-Gly sequence is well suited for PG-surfactants, conferring better extraction efficiency of membrane proteins from biological membranes.



(a) (c) PSI trimer PSII dimer

residual protein from Phycobilisome PSI monomer, PSII monomer

(b)

residual proteins from Phycobilisome

0.6 Absorbance (a.u.)

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0.5 0.4 0.3 0.2

Before CBB stain

0.1

A er CBB stain

0 400


800

Figure 8. (a) Images of the supernatants from thylakoid membrane extractions using buffer D (20 mM HEPES-NaOH (pH 7.2), 10 mM MgCl2, 25% (w/v) glycerol) containing 1 wt% β-DDM, DKDKC12K, DKDKC12D, NPDGC12KK, DPDGC12KK, and AßNp(-)Np(+)AßC12K (from left to right). (b) Absorption spectra of the supernatants extracted from thylakoid membranes using buffer D (20 mM HEPES-NaOH (pH 7.2), 10 mM MgCl2, 25% (w/v) glycerol) containing 1 wt% β-DDM (black line), DKDKC12K (light-green line), DKDKC12D (purple line), NPDGC12KK (red line), DPDGC12KK (yellow line), and AßNp(-)Np(+)AßC12K (blue line). (c) The clear-native PAGE analyses of the extracted fractions from thylakoid membranes using buffer D (20 mM HEPES-NaOH (pH 7.2), 10 mM MgCl2, 25% (w/v) glycerol) containing 1 wt% β-DDM, NPDGC12KK, and DPDGC12KK, before (left) and after (right) CBB staining. Each band was assigned based on the separation profile using 1% β-DDM, which was previously reported.


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Table 3. Extraction efficiencies of surfactants for PSI and PSII from thylakoid membranes. Extraction PG-surfactant

a

Efficiency (%) a

β-DDM

31

DKDKC12K

0

DKDKC12D

0

NPDGC12KK

28

DPDGC12KK

2.4


0

Extraction efficiencies (%) of surfactants were estimated as the ratios of the extracted

Chl a amounts to the total amount of Chl a in thylakoid membranes (0.2 mg of Chl a). Chl a amounts in both fractions were determined by acetone extraction and the molar extinction coefficient at A665 of Chl a in methanol (ε665=79.95 mgChl a-1 L cm-1)).38, 39



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Conclusion We successfully constructed new extraction surfactants for membrane proteins NPDGC12KK based on detail assessments of the candidate sets of PG-surfactants that have β-turn-forming peptides, such as Asn-Pro-Asp-Gly and βAla-Nip(+)-Nip(-)-βAla, as a linker peptide. Using PSI and PSII derived from T. vulcanus as representative membrane proteins, we checked whether NPDGC12KK could maintain the native state of membrane protein structures and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII were damaged upon the use of NPDGC12KK based on detailed photophysical measurements. Using the thylakoid membranes of T. vulcanus as a representative biological membrane sample, we extracted membrane proteins, such as PSI and PSII. Based on an evaluation of extraction efficiency and the maintenance of protein supramolecular structure, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins. Owing to the flexibility in the choice of peptide sequences in PG-surfactants, it is possible to design alternative PG-surfactants with different properties, which could contribute to membrane protein research. Such studies are on going in our group．


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Experimental Procedures Materials (Fmoc)-protected

N-(9-fluorenyloxycarbonyl)

L-amino

acids,

1-hydroxybenzotriazole (HOBT), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

(HBTU),

Rink-amide

AM

resin

(200–400

mesh),

N,N-diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), and N-methylpyrrolidone were purchased from Merck Biosciences (Darmstadt, Germany), Novabiochem (Läufelfingen, Switzerland), and Watanabe Chemical Industries (Hiroshima, Japan). Dichloromethane and methanol (MeOH) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Bromoacetyl bromide, dodecylamine, 3-bromopropionic

acid,

2-(N-morpholino)

ethanesulfonic

acid

(MES),

and

tris(hydroxymethyl)aminomethane (Tris) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Triethylsilane was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Unless otherwise stated, other chemicals and reagents were obtained commercially and used without further purification. Synthesis of PG-surfactants PG-surfactants were synthesized on a Rink-amide AM resin Novabiochem using commercially

available 11, 30

Fmoc-Cys(C12)-OH,

Fmoc-protected

amino

acids,

and

our

synthesized

and Fmoc-protected (+)- and (-)-nipecotic acids.18 For

condensation on a resin, standard condensation reagents (HOBT/HBTU/DIEA) were used. The N-terminus of PG-surfactants was end-capped with Ac2O. After cleavage of the synthesized PG-surfactants from a resin using TFA/H2O = 95/5, the crude PG-surfactants

were

purified

by

reversed-phased

high-performance

liquid

chromatography with a core-shell-type ODS column (Kinetex, Shimadzu, Japan). A linear-gradient of CH3CN and H2O, both including 0.1 vol% TFA, was utilized as an eluent. The purity of each sample was checked by high-resolution ESI-TOF (electrospray ionization time-of-flight) mass spectroscopy. NPDGC12K: HRMS (ESI-TOF, [M + H]+): calcd. for C57H103N12O13S2, 1227.7209; found, 1227.7206 NPDGC12KK: HRMS (ESI-TOF, [M + H]+): calcd. for C63H115N14O14S2, 1355.8159; found, 1355.8157 NPDGC12D: HRMS (ESI-TOF, [M + Na]+): calcd. for C55H95N11O15S2+Na, 1236.6348; found, 1236.6323 25 ACS Paragon Plus Environment


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NPDGC12DD: HRMS (ESI-TOF, [M + Na]+): calcd. for C59H100N12O18S2 + Na, 1351.6618; found, 1351.6609 DPDGC12KK: HRMS (ESI-TOF, [M + H]+): calcd. for C63H114N13O15S2, 1356.7999; found, 1356.7997 AβNp(-)Np(+)AβC12K: HRMS (ESI-TOF, [M + H]+): calcd. for C60H110N11O10S2, 1208.7879; found, 1208.7887. Dynamic light scattering measurements of PG-surfactant assemblies The concentrations of each PG-surfactant in 100 mM Tris HCl buffer (pH 7.0) were set at 0.01, 0.1, and 1 wt% and the mean hydrodynamic diameters of PG-surfactant assemblies for each concentration at 25 °C were estimated using a Zetasizer Nano ZS (Malvern Instruments, Ltd., Malvern, UK). Critical aggregation concentration (CAC) determination for PG-surfactants using 8-anilino-naphtharene-1-sulfonic acid (ANS)31 Fluorescence spectral changes of ANS before and after incorporation into micelle-like assemblies of PG-surfactants in 50 mM phosphate buffer (pH 7) were used to evaluate CACs. From the double linear-fitting analysis for F472, the CAC values of PG-surfactants were evaluated. Purification of PSI derived from T. vulcanus The methods described by Shen and Kamiya,32 Fromme and Witt,33 and Takasaka34 with slight modifications, were used. To solubilize PSI, thylakoid membranes of T. vulcanus (chlorophyll concentration, 2.0 mg-Chl/mL) were incubated with a buffer [20 mM HEPES–NaOH (pH 7.0), 10 mM MgCl2, and 25% (w/v) glycerol] containing 0.6 wt% n-dodecyl-β-D-maltopyranoside (β-DDM) at 0 °C for 30 min in the dark. The extract was separated from the thylakoid membrane by ultracentrifugation (107,000 × g, 60 min). Anion-exchange chromatography (Toyo Pearl DEAE650S; TOSOH, Tokyo, Japan) performed with buffer B (30 mM MES–NaOH (pH 6.0), 5% (w/v) glycerol, 0.03% β-DDM, 3 mM CaCl2, and 0–1000 mM NaCl) at 4 °C was used to isolate the PSI trimer. Increasing the salt concentration to 100 mM led to the elution of PSI monomers and PSII. Increasing the salt concentration to 150 mM NaCl favored trimeric PSI. The trimeric nature and purity of the PSI were confirmed by hrCN-PAGE (high resolution clear native polyacrylamide gel electrophoresis) as described in Latasa et al.35,

36, 37

Subunit composition was analyzed by sodium dodecyl sulfate gel electrophoresis, following Kawakami et al.38 26 ACS Paragon Plus Environment

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Purification of PSII from T. vulcanus The PSII dimer complex was purified from cells of the thermophilic cyanobacterium T. vulcanus grown at 50- 52 °C, as described previously32, 38 Thylakoid membranes isolated from T. vulcanus cells were solubilized with 0.6 wt% (w/v) β-DDM.

The

solubilized

mixture

was

passed

through

an

anion-exchange

chromatography column twice to purify the PSII dimer complex by separation from the PSI and PSII monomer complexes and other proteins. The obtained PSII dimer complex was suspended in 20 mM MES–NaOH (pH 6.0), 20 mM NaCl, 3 mM CaCl2, 5% (w/v) glycerol, and 0.03% β-DDM, and then stored in liquid N2 until use. Replacement of solubilization surfactant via PEG precipitation Solutions of PSI trimers and PSII dimers after purification were diluted with 50% (w/v) PEG1450 (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 17% (w/v) PEG1450. The PSI trimer and PSII dimer precipitates were collected by ultracentrifugation (104,000 × g, 30 min) at 4 °C. The precipitates were washed three times with fresh buffers (in the case of PSI trimers, 40 mM HEPES–NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl2, and 15 mM MgCl2; in the case of PSII dimers, 40 mM MES–NaOH (pH 6.5), 100 mM NaCl, 15 mM CaCl2, and 15 mM MgCl2). The precipitated PSI timer and PSII dimer were resolubilized using 0.1 wt% (w/v) PG-surfactant at 4 °C for 30 min in the dark. Evaluation of photoinduced electron-transfer rate of PSI based on decreases in O2 concentration Measurements of the O2 uptake activity of PSI were conducted at 25 °C using a Clark-type O2 electrode (Hansatech Instruments, DW1, Oxygen Electrode Unit; Norfolk, VA, USA). Red light from a 550-W halogen lamp through a red-pass filter (R-62; Hoya, Saitama, Japan), a heat-cut filter (HA-50, Hoya, Saitama, Japan), and a 12-cm water layer illuminated a 1-mL, 1-cm-diameter reaction vessel. A buffer containing 40 mM HEPES–NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, and 0.4 M sucrose, supplemented with 0.5 mM dichloroindophenol (DCIP) and 2 mM sodium ascorbate as an electron donor couple and 0.5 mM MV2+ as an electron acceptor was used for O2 uptake measurements. An aliquot of sample solution was added to the buffer just before measurements. The concentration of PSI trimer solubilized with 0.006 wt% surfactant was 24 nM. The O2 uptake activity was estimated from the initial negative slope describing the O2 concentration upon illumination. O2 27 ACS Paragon Plus Environment


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uptake from PSI solubilized with β-DDM and PG-surfactants under illumination lasted for approximately 5 min. Flash-induced A698 change of PSI Flash-induced absorbance changes were measured using a split beam spectrophotometer at room temperature.39 The actinic flash was a 5-µs (half-width) pulse from a xenon flash lamp of nearly saturating intensity (300 W). The reaction mixture contained 10 µM DCIP, 1 µM PMS, 10 mM sodium ascorbate, [PSI trimer] = 46 nM, 40 mM HEPES buffer (pH 7.8), 100 mM NaCl, 15 mM MgCl2, and 15 mM CaCl2, at ambient temperature. Signals (100 measurements) were averaged in each case. Evaluation of the photoinduced electron-transfer rate of PSII based on increases in O2 concentration O2 evolution activity of PSII was monitored at 25 °C using a Clark-type O2 electrode. The red-light source was the same as that described above. A buffer containing 40 mM MES–NaOH (pH 6.5), 100 mM NaCl, 15 mM CaCl2, 15 mM MgCl2, and 0.4 M sucrose was used for O2 evolution measurements. An aliquot of a sample solution was added to the buffer just before measurement. The concentration of PSI trimers solubilized with 0.002 wt% surfactant was 24 nM. The exogenous electron acceptor was 0.5 mM phenyl-p-benzoquinone. The O2 evolution activity was estimated from the initial slope of the increase in O2 concentration upon illumination. O2 evolution from PSII solubilized with β-DDM and PG-surfactants under illumination lasted for about approximately 5 min. Fluorescence spectrum of PSI at 77 K The fluorescence spectrum of PSI was recorded using a fluorescence spectrophotometer (FluoroMax; Horiba, Kyoto, Japan) with a laboratory-built liquid N2 Dewar flask at 77 K. PSI dispersed in a buffer (40 mM HEPES–NaOH (pH 7.8), 100 mM NaCl, 15 mM CaCl2, and 15 mM MgCl2) at a concentration of 0.005 mg Chl/mL, which is a low enough concentration to avoid self-absorption, was placed in a sample holder with a light path of 2 mm. The excitation wavelength for the fluorescence measurements was 430 nm. Estimation of the extraction efficiency of the PG-surfactant for PSI and PSII from thylakoid membranes Thylakoid membranes (0.1 mg Chl a) from T. vulcanus were washed three times with 100 µL of buffer C (20 mM HEPES-NaOH (pH 7.2), 10 mM MgCl2) to remove 28 ACS Paragon Plus Environment

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various membrane-bound proteins located at the surfaces of membranes. The resulting thylakoid membrane sample was gently suspended in buffer D (20 mM HEPES-NaOH (pH 7.2), 10 mM MgCl2, and 25% (w/v) glycerol) containing 1 wt% each PG-surfactant and incubated at 4 °C for 0.5 h, followed by centrifugation (15,000 × g) for 35 min at 4 °C to separate extracted fractions. The supernatant, including the extracted PSI and PSII, was separated in a different tube and the extraction efficiencies for PSI and PSII were evaluated by quantification of the Chl a amount in the supernatant (ε665 = 79.95 mgChl a -1L cm-1 in methanol).37, 40 hrCN-PAGE analysis of the extracted PSI and PSII Before the hrCN-PAGE analysi35, 36, 40 of the extracted fractions including PSI and PSII, the solubilization surfactant was exchanged to β-DDM via PEG precipitation using PEG1450. For the clear-native PAGE-analysis, an acrylamide/bis-acrylamide gel was prepared (4% stacking gel, linear 5–12% gradient running gel). The stacking gel was prepared from a 4% solution of acrylamide/bis-acrylamide (37.5/1 (w/w)), containing 25 mM imidazole (pH 7.0) and 0.5 M 6-aminohexanoic acid. The linear 5– 12% gradient gel was prepared by mixing the 5% solution of acrylamide/bis-acrylamide (37.5:1) containing 25 mM imidazole (pH 7.0) and 0.5 M 6-aminohexanoic acid and the 12% solution of acrylamide/bis-acrylamide (37.5:1) containing 25 mM imidazole (pH 7.0), 0.5 M 6-aminohexanoic acid, and 14% glycerol. To initiate polymerization, APS (ammonium persulfate) and TEMED (N, N, N’, N’- tetramethyl-ethylenediamine) were added to each solution. For electrophoresis, a cathode buffer (50 mM tricine, 7.5 mM imidazole, 0.05 % (w/v) Triton X-100, 0.05 % (w/v) deoxycholic acid, and sodium salt), an anode buffer (25 mM imidazole-HCl (pH 7.0)), and 10 µg of Chl a samples of the extracted PSI and PSII mixture was subjected to electrophoresis with a constant current (5 mA) for 12 h. The gel was stained with CBB-250 to visualize each band.



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Author Information Corresponding author *E-mail: [email protected]. Phone: +81-52-735-5237. FAX: +81-52-735-5237

Acknowledgement This work was supported by JSPS KAKENHI (Grant Number 26410177, 15J07454), the Tatematsu Foundation, and in part by the Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers.


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References (1) Gumbiner, B. M. (1996) Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis. Cell 84, 345-357. (2) Lippincott-Schwartz, J., Roberts, T. H., and Hirschberg, K. (2000) Secretory Protein Trafficking and Organelle Dynamics in Living Cells. Annu. Rev. Cell. Dev. Biol. 16, 557-589. (3) Trzaskowski, B., Latek, D., Yuan, S., Ghoshdastider, U., Debinski, A., and Filipek, S. (2012) Action of Molecular Switches in GPCRs – Theoretical and Experimental Studies. Curr. Med. Chem. 19, 1090-1109. (4) Jonckheere, A. I., Smeitink, J. A. M., and Rodenburg, R. J. T. (2012) Mitochondrial ATP Synthase: Architecture, Function and Pathology. J. Inherited Metab. Dis. 35, 211−225. (5) Nelson, N., and Ben-Shem, A. (2004) The Complex Architecture of Oxygenic Photosynthesis. Nat. Rev. Mol. Cell Biol. 5, 971-982. (6) Liu, W., Wacker, D., Gati, C., Han, G. W., James, D., Wang, D., Nelson, G., Weierstall, U., Katritch, V., Barty, A., et al. (2013) Serial Femtosecond Crystallography of G Protein-Coupled Receptors. Science 342, 1521-1524. (7) Seddon, A. M., Curnow, P., and Booth, P. J. (2004) Membrane Proteins, Lipids and Detergents: Not Just A Soap Opera. Biochim. Biophys. Acta 1666, 105-117. (8) Arachea, B. T., Sun, Z., Potente, N., Malik, R., Isailovic, D., and Viola, R. E. (2012) Detergent Selection for Enhanced Extraction of Membrane Proteins. Protein Expr. Purif. 86, 12−20. (9) Koeda, S., Umezaki, K., Sumino, A., Noji, T., Ikeda, A., Yamamoto, Y., Dewa, T., Taga, K., Nango, M., Tanaka, T., et al. (2013) Creation of Cross-Linked Bilayer Membranes That Can Incorporate Membrane Proteins From Oligo-Asp-Based PG-Surfactants. Langmuir 29, 11695−11704. (10) Koeda, S., Umezaki, K., Noji, T., Ikeda, A., Kawakami, K., Kondo, M., Yamamoto, Y., Shen, J.-R., Taga, K., Dewa, T., Ito, S., et al. (2013) Application of Peptide Gemini Surfactants as Novel Solubilization Surfactants for Photosystems I and II of Cyanobacteria. Langmuir 29, 11667−11680. (11) Umezaki, K., Sakai, S., Koeda, S., Yamamoto, Y., Kondo, M., Ikeda, A., Dewa, T., Taga, K., Tanaka, T., and Mizuno, T. (2012) Formation of Planar Bilayer Membranes on Solid Supports Using Peptide Gemini Surfactants. Chem. Lett. 41, 31 ACS Paragon Plus Environment


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1430−1432. (12) Jordan, P., Fromme, P., Witt, H. T., Klukas, O., Saenger, W., and Krauu, N. (2001) Three-Dimensional Structure of Cyanobacterial Photosystem I at 2.5 Å Resolution. Nature 411, 909−917. (13) Umena, Y., Kawakami, K., Shen, J.-R., and Kamiya, N. (2011) Crystal Structure of Oxygen-Evolving Photosystem II at a Resolution of 1.9 Å. Nature 473, 55−60. (14) Searle, M. S. (2001) Peptide models of protein β-sheets: design, folding and insights into stabilizing weak interactions. J. Chem. Soc., Perkin Trans. 2, 1011– 1020. (15) de Alba, E. D., Jimenez, A., and Rico, M. (1997) Turn Residue Sequence Determines β-Hairpin Conformation in Designed Peptides. J. Am. Chem. Soc. 119, 175–183. (16) Zerella, R., Evans, P. A., Ionides, J. M. C., Packman, L. C., Trotter, B. W., Mackay, J. P., and Williams, D. H. (1999) Autonomous Folding of a Peptide Corresponding to the N-terminal β-Hairpin From Ubiquitin. Protein Sci. 8, 1320−1331. (17) Huck, B. R., Fisk, J. D., and Gellman, S. H. (2000) Promotion of Sheet Formation inα-Peptide Strands by a β-Peptide Reverse Turn. Org. Lett. 2, 2607. (18) Chung, Y. J., Huck, B. R., Christianson, L. A., Stanger, H. E., Krauthauser, S., Powell, D. R., and Gellman, S. H. (2000) Stereochemical Control of Hairpin Formation in ß-Peptides Containing Dinipectoric Acid Reverse Turn Segments. J. Am. Chem. Soc. 122, 3995−4004. (19) Menger, F. M., and Keiper, J. S. (2000) Gemini Surfactants. Angew. Chem. Int. Ed. 39, 1906-1920. (20) Vanaken, T., Foxall-Vanaken, S., Castleman, S., and Ferguson-Miller, T. (1986) Alkyl Glycoside Detergents: Synthesis and Applications to study of Membrane proteins. Method. Enzymol. 125, 27-35. (21) Zhao, X., Nagai, Y., Reeves, P. L., Kiley, P., Khorana, H. G., and Zhang, S. (2006) Designer Short Peptide Surfactants Stabilize G-protein-Coupled Receptor Bovine Rhodopsin. Proc. Nat. Acad. Sci. 103, 17707-17712. (22) Hartgerink, J., Beniash, E., and Stupp, S. I. (2001) Self-Assembly and Mineralization of Peptide-Amphiphile Nanofibers. Science 294, 1684-1688. (23) Mullet, J. E., Burke, J. J., and Arntzen, C. J. A (1980) Developmental Study of Photosystem I Peripheral Chlorophyll Proteins. Plant Physiol. 65, 814−822. 32 ACS Paragon Plus Environment

Page 33 of 35

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


(24) Sonoike, K., Hatanaka, H., Katoh, S., and Itoh, S. (1990) Heat-Stability of Iron-Sulfur Centers and P-700 in Photosystem I Reaction Center Complexes Isolated from the Thermophilic Cyanobacterium Synechococcus elongates. Plant Cell Physiol. 31, 865−870. (25) Matsumoto, K., Vaughn, M., Bruce, B. D., Koutsopoulos, S., and Zhang, S. (2009) Designer Peptide Surfactants Stabilize Functional Photosystem-I Membrane Complex in Aqueous Solution for Extended Time. J. Phys. Chem. B 113, 75−83. (26) Tiwari, A., Jajoo, A., and Bharti, S. (2008) Heat-induced Changes in Photosystem I Activity as Measured with Different Electron Donors in Isolated Spinach Thylakoid Membranes. Photochem. Photobiol. Sci. 7, 485−491. (27) Zouni, A., Kern, J., Frank, J., Hellweg, T., Behlke, J., Saenger, W., and Irrgang, K.-D. (2005) Size Determination of Cyanobacterial and Higher Plant Photosystem II by Gel Permeation Chromatography, Light Scattering, and Ultracentrifugation. Biochemistry 44, 4572-4581. (28) Watanabe, M., Iwai, M., Narikawa, R., and Ikeuchi, M. (2009) Is the Photosystem II Complex a Monomer or a Dimer? Plant & Cell Phys. 50, 1674−1680. (29) Watanabe, M., Kubota, H., Wada, H., Narikawa, R., and Ikeuchi, M. (2011) Novel Supercomplex Organization of Photosystem I in Anabaena and Cyanophora paradoxa. Plant & Cell Phys. 52, 162−168. (30) Lumbierrers, M., Palomo, J. M., Kragol, G., Roehrs, S., Müller, O., and Waldmann, H. (2005) Solid-phase Synthesis of Lipidated Peptides. Chem. Eur. J. 11, 7405−7415. (31) De Vendittis, E., Palumbo, G., Parlato, G., and Bocchini, V. A. (1981) Fluorimetric Method for the Estimation of the Critical Micelle Concentration of Surfactants. Anal. Biochem. 115, 278−286. (32) Shen, J.-R., and Kamiya, N. (2000) Crystallization and the Crystal Properties of the Oxygen-Evolving Photosystem II from Synechococcus vulcanus. Biochemistry 39, 14739−14744. (33) Fromme, P., and Witt, H. T. (1998) Improved Isolation and Crystallization of Photosystem I for Structural Analysis. Biochim. Biophys. Acta 1365, 175−184. (34) Takasaka, K., Iwai, M., Umena, Y., Kawakami, K., Ohmori, Y., Ikeuchi, M., and Takahashi, Y. (2010) Structural and Functional Studies on Ycf12 (Psb30) and PsbZ



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 35

Deletion Mutants From a Thermophilic Cyanobacterium. Biochim. Biophys. Acta 1797, 278−284. (35) Wittig, I., Karas, M., and Schägger, H. (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol. Cell Proteomics 6, 1215-1225. (36) Kawakami, K., Umena, Y., Iwai, M., Kawabata, Y., Ikeuchi, M., Kamiya, N., and Shen, J.-R. (2011) Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by deletion mutagenesis and X-ray crystallography. Biochim. Biophys. Acta 1807, 319–325. (37) Latasa, M., van Lenning, K., Garrido, J. L., Scharek, R., Estrada, M., Rodriguez, F., and Zapata, M. (2001) Losses of Chlorophylls and Carotenoids in Aqueous Acetone and

Methanol

Extracts

Prepared

for

RPHPLC

Analysis

of

Pigments.

Chromatographia 53, 385-391. (38) Kawakami, K., Umena, Y., Iwai, M., Kawabata, Y., Ikeuchi, M., Kamiya, N., and Shen, J.-R. (2011) Roles of PsbI and PsbM in photosystem II dimer formation and stability studied by deletion mutagenesis and X-ray crystallography. Biochim. Biophys. Acta 1807, 319–325. (39) Hoshina, S., Sakurai, R., Kunishima, N., Wada, K., and Itoh, S. (1990) Selective Destruction of Iron-Sulfur Centers by Heat/Ethylene Glycol Treatment and Isolation of Photosystem I Core Complex. Biochim. Biophys. Acta 1015, 61−68. (40) Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Determination of Accurate Extinction Coefficients and Simultaneous Equations for Assaying Chlorophylls a and b Extracted With Four Different Solvents: Verification of the Concentration of Chlorophyll Standards by Atomic Absorption Spectroscopy. Biochim. Biophys. Acta 975, 384-394.


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Graphical Abstract Design of new extraction surfactants for membrane proteins from peptide gemini surfactants (PG-surfactants). Masahide Shibata, Shuhei Koeda, Tomoyasu Noji, Keisuke Kawakami, Yuya Ido, Yuichi Amano, Naoki Umezawa, Tsunehiko Higuchi, Takehisa Dewa, Shigeru Itoh, Nobuo Kamiya, Toshihisa Mizuno*


Design of New Extraction Surfactants for Membrane Proteins from

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