Folding and Assembly of Vanilloid Receptor Secondary Structure

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Folding and Assembly of Vanilloid Receptor Secondary Structure Peptide with Hexahistidine Linker at NickelNitrilotriacetic Acid Monolayer for Capsaicin Recognition Koji Nakano, Jun Horiuchi, Shingo Hirata, Makoto Yamanaka, Toshiki Himeno, and Ryoichi Ishimatsu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03202 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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Folding and Assembly of Vanilloid Receptor Secondary Structure Peptide with Hexahistidine Linker at Nickel-Nitrilotriacetic Acid Monolayer for Capsaicin Recognition Koji Nakano*, Jun Horiuchi, Shingo Hirata, Makoto Yamanaka, Toshiki Himeno, and Ryoichi Ishimatsu. Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.

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ABSTRACT

Herein, we report the self-assembly of a synthetic vanilloid receptor (VR) peptide that selectively binds capsaicin. We synthesized a 26-mer peptide—YSEILFFVQS-HHHHHHLAMGWTNMLY (S3HS4) comprising two chemoreceptor domains of transient receptor potential channel (TRPV1) linked by a hexahistidine sequence. High-speed atomic force microscopy (AFM) imaging in water revealed that the peptide structures alternated rapidly between wedge-shaped and linear forms. Circular dichroism spectroscopy showed that 65% of the amide units in the peptide chain adopted -helix structure, which was ascribed to the chemoreceptor domains. S3HS4 developed well-packed monolayers at the Nitreated thiolated nitrilotriacetic acid self-assembled monolayers (SAMs) by chelation of the hexahistidine segment, as characterized by infrared (IR) spectroscopy and AFM that exhibited statistically constant specific height. Therefore, S3HS4 was expected to fold spontaneously upon chelation, and the resulting helix-turn-helix conformers developed films while uniformly oriented: the tilt angle was 69° from the surface normal to the substrate. According to microgravimetric analysis using a quartz crystal microbalance (QCM), the adsorption was 84±47 pmol cm–2 (n = 3), which was almost consistent with the saturation adsorption of an α-helix unit. We also used a QCM to investigate the host– guest reactions of S3HS4, and found that the S3HS4-attached QCM chip bound capsaicin with an apparent binding constant of (4.2±3.6) × 104 M–1 (n = 4), whereas there was no evidence of binding to vanillin or acetophenone. Two controls—a blank chip without


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S3HS4 and a chip modified with a single helical peptide (LAMGWTNMLY-HHHHHH)— produced no capsaicin response. To the best of our knowledge, S3HS4 is the first example of a synthetic VR mimic peptide. We believe that the present surface-directed structurebased design can be used to exploit the α-helix bundle in hexahistidine-linked bishelical peptides.


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INTRODUCTION Membrane proteins are often characterized by bundles of transmembrane (TM) helices that form ligand-gated ion channels (LICs). Transient receptor potential channels, particularly subfamily V member 1 (TRPV1), are among the most studied LICs.1–7 Interestingly, TRPV1 becomes excited in response to pain-causing pungent substances.1, 5—6

It has therefore elicited unprecedented efforts to develop pain relief medication and

chemical pain sensors.8 Mammalian cell expression cloning methods have successfully produced the receptor protein for research, but the typical challenges of improving affinity, specificity, and stability are becoming increasingly important. Moreover, the practical application of receptor proteins requires economical mass production. The development of vanilloid receptor mimics will provide an alternative to recombinant protein expression, thereby facilitating future breakthroughs. Currently, solid-phase peptide synthesis (SPPS) is the primary tool for preparing diverse peptides; regardless of whether they are naturally-occurring or artificially-devised, researchers can synthesize various protein fragments with quite large numbers of amino acid residues.9 As a result, synthetic peptides have proven useful for studying ligand– protein and protein–protein interactions. Biological studies on LICs have produced some unique results. With regard to TRPV1, mutant inactivation experiments have identified the amino acids that are essential for chemoreception,6 and cryo-electron microscopy has revealed its 3D structure.10 The results indicated that only two of the six TM helices contain


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key amino acid residues, i.e., the 3rd TM segment with Y511 and the 4th TM segment with M547, W549, and T550. They are brought into close proximity to form the guestrecognition pocket by protein folding. Accordingly, if it were possible to synthesize a corresponding pair of peptides and present them in the required arrangement, an artificial receptor molecule that could mimic the structure and/or function of the vanilloid receptor could be obtained. Previously, we reported a synthetic chemoreceptor domain of TRPV1, LAMGWTNMLY (S4); fluorescence spectroscopy measurements in homogeneous solution showed that the peptide had a certain affinity for vanillin. We also synthesized a fusion peptide, LAMGWTNMLY-HHHHHH (S4H), for use in heterogeneous systems including biosensors/biodevices.11 Certain types of -helical peptides containing a sulfur function that promotes gold–sulfur interaction, e.g., cystine,12 lipoic acid,13 alkyl-mercaptans14–16, and alkyl-mercaptides,17,

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develop self-assembled monolayers (SAMs). Similarly, with

bioconjugation purposes, previous researches so far have shown that a hexahistidine segment is also an efficient anchor if the corresponding peptide is directed to Ninitrilotriacetic acid (NTA)-treated surfaces.19–22 Regarding the S4H-immobilization, surface-processed transducers are responsive to vanillin with a given selectivity. In the present study, we aimed to expand current single-constituent SAMs—developed by ourselves and others—to a binary format; we combined a synthesized pair of chemoreceptor domains with a hexahistidine sequence: YSEILFFVQS-HHHHHH-


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LAMGWTNMLY (S3HS4). Hexahistidine coordinates to the surfaces of Ni complexes with vacant coordination sites so that the chelation could lead to the formation of a trisegmented peptide with a helix-turn-helix tertiary structure. This structure brings the chemoreceptor domains into close proximity, thereby recreating the guest-recognition domain found in TRPV1. High-speed atomic force microscopy (AFM) imaging and circular dichroism (CD) spectroscopy revealed the tertiary structure of the peptide in homogeneous solution. Several surface analysis techniques including AFM, Fouriertransform infrared spectroscopy (FT-IR), and the application of a quartz-crystal microbalance (QCM) have enabled the detailed characterization of binary-peptide SAMs. Interestingly, we found that S3HS4 SAMs have considerable affinity for capsaicin—an active component of chili peppers that produce the sensation of pain. We used QCM to demonstrate the potential use of S3HS4 in a chemical pain sensor.

EXPERIMENTAL SECTION Synthesis of S3HS4 and SNTA. All chemicals were guaranteed reagents and were used as received. The peptides were prepared in their N-acetylated forms by Fmoc solid phase peptide

synthesis

(SPPS).

(1S)-N-[5-[(4-mercaptobutanoyl)amino]-1-

carboxypentyl]iminodiacetic acid (SNTA) was synthesized according to the method described in the literature.20 (Supporting Information).


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Preparation of SAMs. We used gold (Au) films deposited on three different substrates. Each gold-coated substrate was first chemically etched using piranha solution (Caution! Piranha solution is a strong oxidizing agent and extreme care is necessary). The cleaned substrate was immersed in aqueous 10 mM SNTA solution for 24 h at room temperature (RT), typically 22–25 °C. It was then subjected to Ni2+ chelation for 1 h at RT by casting 100 L of aqueous 5 mM NiCl2 solution onto the surface. Similarly, the peptide was immobilized by exposing the Ni-treated SNTA/Au to 100 L of peptide solution (5 mM in 50% DMSO/TE buffer (DMSO is dimethyl sulfoxide; TE is Tris, a common pH buffer, and ethylenediaminetetraacetic acid (EDTA)) for 3 h at RT. The substrate was rinsed thoroughly with deionized water and dried under vacuum for 1 h as necessary. Sample preparations including subsequent measurements were carried out at ambient temperature, typically 22–25 °C. AFM. Liquid-phase AFM imaging was carried out using a high-speed atomic force microscope (Research Institute of Biomolecule Metrology (RIBM) Co., Ltd., Tsukuba, Japan) operated in intermittent contact mode. The detailed measurement conditions including sample preparation were as previously reported.23 The AFM observations in the corresponding immobilized state were made using a JSPM 5410 Scanning Probe Microscope (JEOL Ltd., Tokyo Japan) operated in a vacuum in non-contact mode with frequency modulation detection.11 Gold films comprising crystals with (111) structure (111 indicates the Miller indices; Agilent Technologies Inc., Santa Clara, CA, USA) were used as substrates, and were treated using the same procedure described above.


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Spectroscopy measurements. Circular dichroism spectra were acquired on a J-1500 spectropolarimeter (JASCO Co., Tokyo, Japan). The sample solutions were placed in standard quartz cuvettes (10-mm light path), and the instrument including the sample chamber was purged with N2 throughout the experiments. The IR spectra were obtained using a JASCO FT-IR 620/V spectrometer. The transmittance spectra for the sample peptide were obtained using the KBr pellet method. The SNTA-Ni SAMs were developed on polycrystalline Au films resistively deposited onto TEMPAX Float® glass-plates (Kinoene Kogaku, Tokyo, Japan). Reflection–absorption (RA) spectra were measured with 80° angles of incidence using a polarizer to eliminate s-polarized light. Typically, 256 scans at 4 cm–1 resolution were collected under vacuum conditions. QCM. A QCA 934 quartz crystal resonator biosensing system (SEIKO EG&G Co. Ltd., Tokyo, Japan) was used with commercially available Au quartz crystals (AT-cut, 9 MHz). The tips were first etched with piranha solution (caution! Piranha solution is a strong oxidizing agent and extreme care is necessary), then mounted in an appropriately shaped polytetrafluoroethylene (PTFE) cell so that the Au surface was exposed (0.20 cm2). The measurement was carried out according to the method described in a previous report.11

RESULTS AND DISCUSSION Solution structure characterized by AFM and CD. To prepare the synthetic vanilloid receptor, we simply copied both the chemoreceptor domains—which are sequentially


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discontinuous and distant in TRPV1—into a continuous stretch of peptide chain with an interconnecting hexahistidine sequence. This approach was consistent with structurebased design,24 which is a useful method of developing a protein-binding site mimic consisting of two or more recognition segments. The proposed 3D structure of TRPV16 suggests that S3HS4 consists of two rigid, rod-like segments interconnected by a flexible, rope-like segment. AFM imaging of the peptide in aqueous solution successfully characterized the structure by visualizing each peptide molecule. As Figure 1 illustrates, the surface of the S3HS4-treated mica sheet was covered with dozens of nanometer-sized substances. The apparent density increased when the sample was prepared using more S3HS4 (data are not shown); we presumed that the surface bodies were the peptide molecules. The AFM image indicates that most of the surface bodies were wedge-shaped and had a typical length of 7 nm, although some had a linear conformation. Protein structures fluctuate in conformational basins. Such temporal changes to the peptide morphology are obvious from the animated AFM images; the conformation of S3HS4 changed rapidly and ceaselessly over time (Supplementary information). A simple histogram analysis conveniently and quantitatively characterized the tertiary structure of S3HS4 (Figure 1B). Here, we assumed an adsorption model in which the major molecular axis oriented parallel to the substrate surface. The peptide molecules had a specific height of 0.7 nm, which was considerably smaller than the diameter of the -helix expressed by the mean side chain set (1.2 nm), but somewhat


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larger than the -helix core. Therefore, it is possible to speculate that adsorption of the peptide caused deformation of the side chains in the helical structure. The frequency distribution profile that traced a typical normal distribution was further analyzed by nonlinear curve fitting. When we assumed a set of Gaussian function peaks, the results showed that the morphology specified a two-component model. Specifically, one component was a heavily-textured element and the other was a slightly higher one with a highly dispersed full width at half maximum (FWMH) of 0.18. The former component appeared to have a larger areal fraction (0.7), which can be attributed to the -helix units adopted by 20 amino acids out of 26. In contrast, the weaker component (areal fraction 0.3) can be ascribed to the hexahistidine sequence; the peptide segment is flexible and adopts various conformation, and therefore its height changes. We also investigated the tertiary structure of S3HS4 in bulk using molecular spectroscopy. Figure 2 shows a representative CD spectrum of the peptide dissolved in trifluoroethanol (TFE). There are two negative shoulders at 222 and 208 nm, and an intense positive band at 192 nm, all of which are typical of polypeptide α-helix structures. We also examined the spectral data on the DICHROWEB server.25 This procedure preforms spectral deconvolution, providing calculated secondary structure contents by referencing CD spectral databases. We obtained a set of data for five typical structural components that shape S3HS4: 44% -helix, 21% disordered helical components, 2% -sheet, 3% disordered sheet components, 12% turn components, and 18% unordered structural


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components. Therefore, the primary component includes two types of -helix, which presumably resulted from the TM3 and TM4 sequences. In contrast, the hexahistidine sequence with neighboring amino acids preferred either the turn or the unordered structures. It should be stressed that the results of content determination for each structural unit in bulk agreed well with the individually measured average values.

Immobilization chemistry characterized by AFM, IR, and QCM. First, we used noncontact-mode AFM imaging to characterize the immobilization/folding strategy. The results of molecular imaging were basically similar to those obtained in solution, but the surface state of the sample was markedly different (Figure 3). The substrate surface was completely and almost uniformly covered with countless surface bodies with shapes and sizes similar to those found in solution. There was no local order in the AFM images of the non-S3HS4 treatment substrate or the naked Au. Once more, each surface body was considered to be an S3HS4 molecule bound to a Ni site in the SAMs. However, the frequency distribution profile determined the statistical height of the peptide to be 1.3 nm, which indicates the monomolecular adsorption of S3HS4. The height was slightly larger than the specific diameter of an -helix. Tight and compact packing of the peptide molecules may lead to the upthrust of both molecular termini out of the outermost region of the substrate.


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Next, we qualitatively analyzed the film using IR spectroscopy (Figure 4). There are two characteristic peaks at 1659 cm–1 and 1538 cm–1 in the reflection–absorption (RA) spectrum, which can be ascribed to the amide I and amide II bands, respectively. Furthermore, there is a peak at 1230 cm–1, which can be attributed to amide III absorption. Therefore, the primary component of the film was identified as consisting of peptide molecules, more specifically S3HS4. The absorbance ratio between amide I and amide II provides quantitative information about the molecular orientation.26 We examined S3HS4 chelation based on the following equation:27

[12(3cos 𝛾 ― 1)][12(3cos 𝜃 ― 1)] + 1 [12(3cos 𝛾 ― 1)][12(3cos 𝜃 ― 1)] + 1

1 (2𝑆 𝑆 + 1) 𝐶 1 𝐼1 3 H 𝑡1 𝐷obs = = =𝐶 𝐼2 1 𝐶2 (2𝑆𝑡2 + 1) 3

2

2

2

2

1

(1).

2

Ii, Ci, SH, and Sti represent the observed absorbance, the proportionality constant, the order parameter of the helix axis, and the order parameter of the transition moments, respectively. I1 and I2 correspond to amide I and amide II, respectively. Likewise, Dobs, , i, and C stand for the observed amide I/amide II absorbance ratio, the tilt angle of the helix axis from the normal surface, the angle between the transition moment (amide I or amide II) and the helix axis, and the scaling constant, respectively. The amide I/amide II absorbance ratio for the S3HS4 film—obtained by comparison of the corresponding peak area—was 1.56. Similarly, the scaling factor was obtained from the amide I/amide II


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absorbance ratio for S3HS4 in a KBr pellet taken at a random orientation: 2.64 in the present case (Figure S3). The angles of the transition moments from the helix axis—1 and 2—were 39° and 75°, respectively, as reported in the literature.27 Simple arithmetic using Eq. (1) produced a tilt angle of 69°. The authors of a previous study reported that the tilt angle for lipoic acid-derived peptides varied from 30° to 66°.27 Our result was a slightly larger, but was still very similar to the literature value. Because S3HS4 films are uniform in the corresponding AFM images, the -helix unit is also considered welloriented in the corresponding adsorbed state. Polyhistidine tags have been used by introducing at either end of polypeptide chain so far, but we used it as a bridge connecting a set of helical segments. At the moment, a clear experimental fact showing Ni-chelation has not been obtained, but our chelation strategy, when applied to binary adsorbates such as S3HS4, is very likely to promote the formation of a multi-helix bundle fold at the substrate surfaces. Helical peptides are often involved in biological molecular recognition events; examples include enzymatic reactions and various DNA-binding proteins such as the zinc finger, the helix-turn-helix, and the leucin zipper.28 Helical peptides are also involved in various enzyme models.29,30 The precise display of the helical peptide unit is common in these examples; it necessarily requires the rational design of incorporated structure-forming units, e.g., linker a peptides, hinge groups, and synthetic rigid molecules. However, we successfully prepared a hexahistidine-linked bishelical peptide with a straightforward


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design. The chelation reaction folded the peptide simply and directly into a multi-helix bundle, as intended. Quantitative curve fitting analysis for the IR-RAS data revealed that the amide I band gave roughly six-component peaks including 1698 cm-1 (antiparallel sheet), 1683 cm-1 (-turn), 1668 cm-1 (310-helix), 1652 cm-1 (-helix), 1636 cm-1 (-sheet), and also 1619 cm-1 (-sheet) (Figure S4). Quantification of the specific peak area revealed that the helix content reached up to 45%, which was almost consistent to that of CD measurements. Moreover, neither of the characteristic peak for random coil (typically 1646 cm-1) nor that for aggregated strands (typically 1611 cm-1) was noticed in the spectrum.31 These results showed that S3HS4 can stably bind to the SNTA SAMs, but still retains

most

of

the

helical

structure

without

noticeable

denaturation.

This

assembly/folding approach is a new structure-based design strategy for functional peptides.24 Finally, we conducted QCM analysis on the chelation reaction between S3HS4 and the Ni site in the SAM. First, we observed a specific resonant frequency decrease that was equivalent to 181±22 pmol cm–2 (n = 3) for SNTA chemisorption (Figure S5). The obtained data were rather small compared with the theoretical data for alkanethiol (760 pmol cm– 2).

The sterically bulky aminopolycarboxyl group might hinder molecular association at

the surface, but previous AFM study showed that SNTA developed well-organized SAMs.11 During the chelation of S3HS4, the resonance frequency decreased further (Figure 5), eventually reaching equilibrium at a specific binding amount of 84±47 pmol cm–2 (n = 3),


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which was considerably smaller than that of SNTA at the surface. However, a reciprocal αhelix cross-section (1.1 nm2) limits the theoretical surface concentration for a bishelical adsorbate at a maximum below 73 pmol cm-2. Considering the AFM and IR data, the chelation strategy of hexahistidine-linked bishelical peptides such as S3HS4 can produce well-packed monolayers when subjected to SNTA-Ni SAMs.

QCM studies of host–guest reaction between S3HS4 and capsaicin. The experimental results obtained here have indicated that S3HS4 spontaneously develops a multi-helix bundle fold via chelation. Therefore, we attempted to investigate the spatial structure necessary for chemoreception; a molecular building model provided detailed information about the guest-binding site (Figure 6). In the expected structure, three residues—Y1, M19, and T22—are in close proximity, centered on W21. This allows the peptide to present an exposed hydrophobic cavity towards the external solution phase, enabling host–guest interaction. Preliminary experiments revealed that S3HS4 was less soluble in typical hydrophilic solvents. Therefore, QCM measurements using receptor-modified chips were used to study host–guest reactions. The QCM chip used in the experiments illustrated in Figure 5 was subsequently used for guest-binding reactions. Figure 7A shows the time course of the oscillating frequency change when the chip was exposed to 100 µM vanillin solution. We found that after the first sharp convergence, the oscillation frequency was almost the same as the initial value.


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The chip was subsequently exposed to acetophenone in a similar way, and again no change was noticed (Figure 7B). When the same experiment was conducted using capsaicin solution, the chip responded for the first time (Figure 7C). The frequency change (13 Hz) was equivalent to 47 pmol cm–2 capsaicin, which reached 81% binding of S3HS4 at the SAM surface (58 pmol cm–2). Repeated measurements using newly prepared S3HS4 chips showed that the sensor responded to capsaicin with good reproducibility (24±12 Hz, n = 5). A blank chip without the host peptide was used as a control; the resonance frequency fluctuated slightly and periodically after injecting capsaicin, but there was no clear frequency change (Figure 7D). A series of measurements revealed that the S3HS4attached chips responded to capsaicin with considerable selectivity. The capsaicin response was further analyzed based on a 1:1 binding mechanism (Eq. 2). Here, the concentration of free capsaicin can be assumed to be constant (Ccapsaicin = 100 µM):

𝐾𝑎𝑝𝑝 =

[𝐒𝟑𝐇𝐒𝟒 ∙ capsaicin]𝑆 [𝐒𝟑𝐇𝐒𝟒]𝑆 × [capsaicin]

=

[𝐒𝟑𝐇𝐒𝟒 ∙ capsaicin]𝑆

{𝐶𝐒𝟑𝐇𝐒𝟒, 𝑠 ― [𝐒𝟑𝐇𝐒𝟒 ∙ capsaicin]𝑠} × 𝐶capsaicin

(2)

Where subscript s denotes the immobilized state and C represents the total concentration of the corresponding chemical species. Repeated experiments with various QCM-chip preparations revealed that capsaicin bound 78±19% (n = 5) of the surface-attached S3HS4, from which was obtained an apparent binding constant of (4.2±3.6) × 104 M–1. A


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previous report showed that S4H, a single-helix host, selectively bound vanillin with an apparent binding constant of 2.7 × 103 M–1.11 For comparison, we investigated the capsaicin response at an Au-chip with a HS4/SNTA/Au interfacial structure, but no frequency response was confirmed (Figure 7E). Introducing an additional counterpart helix strengthened the binding of the vanilloid moiety by 16 times, and may have increased the selectivity of the vanilloid receptor with regard to capsaicin. The 3D model of S3HS4 hints that, unlike S4H where its binding site is completely exposed, the chemoreception site is too tight to form corresponding host-guest complexes; during the reaction the receptor needs to change the conformation to some extent. In other words, unless the destabilizing energy is compensated, the host-guest reaction occurs within only a limited extent. Increasing the number of helices may further increase the performance of the chemoreceptor. Modification of amino acid residues at the chemoreceptor site is also future task. Our totally synthetic approach allows us chemical mutation experiments, which will be useful in elucidating the chemoreception mechanism of VRs. The research is continuing and the results will be reported elsewhere. Several high-performance liquid chromatography (HPLC) fluorescence detection methods have been used to determine the amounts of capsaicin and members of the capsinoid family in chili peppers.33 However, such methods often require extensive labor and expensive instruments, and the experiments tend to be time-consuming. Several voltammetric techniques34–38 and impedance analysis39 are also under review with regard


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to items such as cost, ease of use, and sensitivity. They rely on various advanced materials—e.g., carbon nanotubes, boron-doped diamonds, nanoparticles, and graphene—for electrode surface modification. Therefore, our artificial vanilloid receptor would also be suitable for high-performance electrochemical capsaicin sensing. The results will be reported elsewhere.

SUMMARY AND CONCLUSIONS Molecular recognition by -helix domains on protein surfaces is ubiquitous in vivo. This type of interaction motif is of great importance in the recapitulation of natural systems by synthesizing artificial mimetic peptides. Previous research has mainly been conducted using single-helix constituents. Interestingly, however, recent studies have revealed that several multicomponent helix bundles function as enzymes.40 Similarly, with regard to immobilized systems, the surface constituents are limited to single-component systems.12,41 In the present study, we successfully developed a hexahistidine chelation method for peptide SAMs that uses bishelical bundle structures. By reflecting the chemoreceptor domain of TRPV1 in the amino acid sequence, the resulting vanilloid receptor mimic exhibited host–guest reactivity, as intended. To the best of our knowledge, S3HS4 is the first example of a synthetic capsaicin receptor. Currently, structure-based design24 is the primary approach to obtaining molecular recognition protein mimics comprising several structural units such as helices, sheets, and turns. Our strategy uses


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structure-based design at substrate surfaces. The method will provide a versatile means by which hexahistidine-linked peptides can exert a particular function characteristic of a helix-turn-helix folded structure.

SUPPORTING INFORMATION We have provided: pdfs describing the synthetic procedure and analysis data for S3HS4 and SNTA (Figure S1 and Figure S2); the IR spectra for S3HS4 measured using the KBr palette method (Figure S3); summary of quantitative curve fitting analysis for the amide I band (Figure S4); a time-course for the QCM analysis of SNTA SAMs and Ni-treatment (Figure S5). An animated version of the AFM images measured in liquid is also available as a .docx file (Figure S6). The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding Author: Koji Nakano *Telephone: +81928022890; e-mail: [email protected] ORCID Koji Nakano: 0000-0002-4860-5389


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Notes The authors declare no competing financial interest. Author Contributions K.N. conceived the project, designed the methods, analyzed the results, performed the computer modeling, and wrote the manuscript. J.H. performed the synthetic work, and acquired the CD, IR, and QCM data. S.H. performed the initial peptide syntheses including that of SNTA. M.Y. and T.H. performed the AFM imaging of S3HS4 in the corresponding SAM state and in solution, respectively. R.I. assisted with the validation of the data. All authors have given their approval for publication. Funding Sources We received funding from the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number JP25620115 and JP18H03782).

ACKNOWLEDGMENTS We thank Frank Kitching, MSc., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.


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(20) Roure, O. D.; Debiemme-Chouvy, C.; Malthete, J.; Silberzan, P. Functionalizing Surfaces with Nickel Ions for Grafting of Proteins. Langmuir 2003, 19, 4138—4143. (21) Huang, Z.; Park, J. I.; Watson, D. S.; Hwang, P.; Szoka, Jr. F. C. Facile Synthesis of Multivalent Nitrilotriacetic Acid (NTA) and NTA Conjugates for Analytical and Drug Delivery Applications. Bioconjugate Chem. 2006, 17, 1592−1600. (22) Baio, J. E.; Cheng, F.; Ratner, D. M.; Stayton, P. S.; Castner, D. G. Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondaryion mass spectrometry. J Biomed. Material Res. 2011, 97A, 1—7. (23) Tanabe, J.; Nakano, K.; Hirata, R.; Himeno, T.; Ishimatsu, R.; Imato, T.; Okabe, H.; Matsuda, N. Totally synthetic microperoxidase-11. Royal Soc. Open Sci., 2018, 5, 172311—172320. (24) Groß, A.; Hashimoto, C.; Sticht, H.; Eichler, J. Synthetic Peptides as Protein Mimics. Front. Bioeng. Biotechnol. 2016, 3, 1—16. (25) Whitmore, L.; Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res., 2004, 32, W668—W673. (26) Boncheva, M.; Vogel, H. Formation of Stable Polypeptide Monolayers at Interfaces: Controlling Molecular Conformation and Orientation. Biophys. J. 1997, 73, 1056—1072.


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(27) Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J. Formation of oriented helical peptide lyers on a gold surfaces due to the self-assembling properties of peptides. Langmuir 1998, 14, 6935—6940. (28) Harrison, S. C. A structural taxonomy of DNA-bindig domains. Nature, 1991, 353 715—719. (29) Lombardi, A.; Nastri, F.; Pavone, V. Peptide-Based Heme−Protein Models. Chem. Rev., 2001, 101, 3165—3189. (30) Reedy, C. J.; Gibney B. R. Heme Protein Assemblies. Chem. Rev., 2004, 104, 617— 649. (31) Baginska, K.; Makowska, J.; Wiczk, W.; Kasprzykowski, F.; Chmurzynski, L. Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy. J. Peptide Sci., 2008, 14, 283—289. (32) Spiga, O.; Bernini, A.; Scarselli, M.; Ciutti, A.; Giovannoni, L.; Laschi, F.; Bracci, L.; Niccola, N. NMR studies on Ni(II) induced cyclization of a histidine-tagged peptide. J. Peptide Sci. 2002, 8, 634—641. (33) Barbero, G. F.; Liazid, A.; Palma, M.; Barroso, C. G. Fast determination of capsaicinoids from peppers by high-performance liquid chromatography using a reversed phase monolithic column. Food Chemistry, 2008, 107, 1276—1282.


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Figure Captions Figure 1. Representative atomic force microscopy (AFM) images of S3HS4 in deionized water (A: 500 nm × 500 nm). For experiments, we necessary used a set of different samples because of unstable measurments. The inset is an enlarged image showing the wedgeshaped conformers and the linear molecules. Panel B represents plots of frequency versus specific height (filled circles). The two thin lines represent the results of nonlinear curve fitting analysis, which reproduced the experimental results well if simply summed (thick line).

Figure 2. Circular dichroism (CD) spectrum for 31 µM S3HS4 dissolved in trifluoroethanol (TFE) at 25 °C. Theoretical CD intensity (filled circles) calculated for a set of five typical components—i.e., 44% -helix, 21% disordered helical components, 2% -sheet, 3% disordered sheet components, 12% turn components, and 18% unordered structural components—reproduced the experimental data (solid line) well.

Figure 3. Typical surface topographic images for an Au (111) substrate with an S3HS4/SNTA-Ni/Au interfacial structure (A: 250 nm × 250 nm), and a magnified view of the marked region (B). Panel C represent plots of frequency versus specific height. The


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obtained data set (filled circles) traces a typical normal distribution representing a single Gaussian function peak (solid line).

Figure 4. A representative infrared reflection–absorption (IR-RA) spectrum of an S3HS4 film at the thiolated nitrilotriacetic acid (SNTA)-Ni self-assembled monolayer (SAM). The inset shows the results of peak-fitting analysis for the amide I and amide II regions (thin lines); the overlapping two amide bands were peak-separated by applying a Gaussian function and their sum total (thick line) precisely reproduces the original data (filled circles). The ratio of each peak area thus obtained was taken and used for the later calculation: the amide I/ amide II ratio to be 1.56.

Figure 5. A representative time-course of resonant frequency change during the chelation reaction between S3HS4 and the Ni site in the thiolated nitrilotriacetic acid (SNTA) selfassembled monolayers (SAMs). The reaction was initiated using 31 µM S3HS4 solution in 20% DMSO/pH 7 Tris at the elapsed time indicated by the arrow.

Figure 6. A 3D model for an optimized structure of S3HS4 obtained by density functional theory (DFT) calculation (G16/B3LYP) displaying the solvent accessible surface. The three


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types of fragments—a set of helical domains and a hexahistidine sequence—were first independently optimized, and the combined 26-mer was further optimized in a similar manner. With regard to hexahistidine, the lowest energy conformer of a model peptide32 was used as the initial structure. A cis isomer of capsaicin is shown for comparison.

Figure 7. Typical time-courses of resonant frequency changes of the S3HS4-Au chip after injection of (A) vanillin, (B) acetophenone, and (C) capsaicin. In (D) and (E), similar timecourses with regard to capsaicin were obtained with a blank Au chip and the S4H-Au, respectively. The reactions were initiated using each substance (100 µM) at the times indicated by the arrows.


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A

B

30 nm

Figure 1. Representative atomic force microscopy (AFM) images of S3HS4 in deionized water (A: 500 nm × 500 nm). The inset is an enlarged image showing the wedge-shaped conformers and the linear molecules. Panel B represents plots of frequency versus specific height (filled circles). The two thin lines represent the results of nonlinear curve fitting analysis, which reproduced the experimental results well if simply summed (thick line).


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Figure 2. Circular dichroism (CD) spectrum for 31 µM S3HS4 dissolved in trifluoroethanol (TFE) at 25 °C. Theoretical CD intensity (filled circles) calculated for a set of five typical components—i.e., 44% -helix, 21% disordered helical components, 2% -sheet, 3% disordered sheet components, 12% turn components, and 18% unordered structural components—reproduced the experimental data (solid line) well.


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B

A

C

Figure 3. Typical surface topographic images for an Au (111) substrate with an S3HS4/SNTA-Ni/Au interfacial structure (A: 250 nm × 250 nm), and a magnified view of the marked region (B). Panel C represent plots of frequency versus specific height. The obtained data set (filled circles) traces a typical normal distribution representing a single Gaussian function peak (solid line).


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Figure 4. A representative infrared reflection–absorption (IR-RA) spectrum of an S3HS4 film at the thiolated nitrilotriacetic acid (SNTA)-Ni self-assembled monolayer (SAM). The inset shows the results of peak-fitting analysis for the amide I and amide II regions (thin lines); the overlapping two amide bands were peak-separated by applying a Gaussian function and their sum total (thick line) precisely reproduces the original data (filled circles). The ratio of each peak area thus obtained was taken and used for the later calculation: the amide I/ amide II ratio to be 1.56.


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S3HS4

Figure 5. A representative time-course of resonant frequency change during the chelation reaction between S3HS4 and the Ni site in the thiolated nitrilotriacetic acid (SNTA) selfassembled monolayers (SAMs). The reaction was initiated using 31 µM S3HS4 solution in 20% DMSO/pH 7 Tris at the elapsed time indicated by the arrow.


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S3

S4 Y26

Y1 T22 W21 M19

capsaicin

H16 H11

Figure 6. A 3D model for an optimized structure of S3HS4 obtained by density functional theory (DFT) calculation (G16/B3LYP) displaying the solvent accessible surface. The three types of fragments—a set of helical domains and a hexahistidine sequence—were first independently optimized, and the combined 26-mer was further optimized in a similar manner. With regard to hexahistidine, the lowest energy conformer of a model peptide31 was used as the initial structure. A cis isomer of capsaicin is shown for comparison.


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A

B

C

D

E

Figure 7. Typical time-courses of resonant frequency changes of the S3HS4-Au chip after injection of (A) vanillin, (B) acetophenone, and (C) capsaicin. In (D) and (E), similar timecourses with regard to capsaicin were obtained with a blank Au chip and the S4H-Au, respectively. The reactions were initiated using each substance (100 µM) at the times indicated by the arrows.


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TOC image


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Folding and Assembly of Vanilloid Receptor Secondary Structure

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