Synthesis and Self-Assembly of His-tag Hybrid of Substrate-Binging Short Domain in Transient Receptor Potential Vanilloid Type 1 for Vanillin Sensing Application

Abstract

The amino acid sequence of vanillin-binding site of transient receptor potential vanilloid type 1 from rat, Leu 544 -Tyr 553 , was extracted and hybridized with His-tag. The hexadecamer invariant chain peptide, Leu-Ala-Met-Gly-Trp-Thr-Asn-Met-Leu-Tyr-His-His-His-His-His-His (VBH), was prepared by solid-phase peptide synthesis. Circular dichroic spectral measurements determined the α-helix content to be 17%, which was consistent to that of short peptides. In a combined use of thiol-derivatized nitrilotriacetic acid (s-NTA) monolayers, the His-tag successfully attached the whole peptide on gold substrate surfaces through Ni 2+ -chelation (Γ VBH = 224 ± 120 pmol cm -2 , n = 8). Moreover, various surface analyses including atomic force microscopy imaging, FT-IR spectroscopy, and quartz-crystal microgravimetry (QCM) revealed self-assembly (SA) of VBH at the S-NTA monolayer surfaces. QCM measurements also showed that vanillin, the major component of natural vanilla flavoring, binds to VBH SAs (K app = 2.7 × 10 3 M -1 ). The affinity of host-guest binding remains limited but possesses a certain degree of selectivity; for cases of structural analogs that give a pleasant flavor, acetophenone showed rather weak affinity (K app = 2.8 × 10 2 M -1 ) whereas 4-heptanone did not bind at all. With these results VBH was concluded to be useful in vanillin sensing as a supramolecular affinity host.

Folding and Assembly of Vanilloid Receptor Secondary-Structure Peptide with Hexahistidine Linker at Nickel–Nitrilotriacetic Acid Monolayer for Capsaicin Recognition

“…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 . During the chelation of S3HS4 , the resonance frequency decreased further (Figure ), eventually reaching equilibrium at a specific binding amount of 84 ± 47 pmol cm –2 ( n = 3), which was considerably smaller than that of SNTA at the surface.…”

“…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) × 10 4 M –1 . A previous report showed that S4H , a single-helix host, selectively bound vanillin with an apparent binding constant of 2.7 × 10 3 M –1 . 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 E).…”

“…The detailed measurement conditions including sample preparation were as previously reported . 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 the noncontact mode with frequency modulation detection . Gold films comprising crystals with (111) structure (111 indicates the Miller indices; Agilent Technologies Inc., Santa Clara, CA) were used as substrates and treated using the same procedure described above.…”

“…Piranha solution is a strong oxidizing agent and extreme care is necessary) and then mounted in an appropriately shaped poly­(tetrafluoroethylene) cell so that the Au surface was exposed (0.20 cm 2 ). The measurement was carried out according to the method described in a previous report …”

“…Previously, we reported a synthetic chemoreceptor domain of TRPV1, LA M G WT NMLY ( S4 ); fluorescence spectroscopy measurements in homogeneous solution showed that the peptide had a certain affinity for vanillin. We also synthesized a fusion peptide, LA M G WT NMLY-HHHHHH ( S4H ), for use in heterogeneous systems including biosensors/biodevices . Certain types of α-helical peptides containing a sulfur function that promotes gold–sulfur interaction, e.g., cystine, lipoic acid, alkyl-mercaptans, − and alkyl-mercaptides, , develop self-assembled monolayers (SAMs).…”