Synthesis and Self-Assembly of His-tag Hybrid of Substrate-Binging Short Domain in Transient Receptor Potential Vanilloid Type 1 for Vanillin Sensing Application
Koji Nakano, Shingo Hirata, Jun Horiuchi, Ryoichi Ishimatsu, Toshihiko Imato, Takeshi Onodera, Kenshi Hayashi
Transactions of the Materials Research Society of Japan
The amino acid sequence of vanillin-binding site of transient receptor potential vanilloid type 1 from rat, Leu544–Tyr553, 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 Ni2+-chelation (ΓVBH = 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 (Kapp = 2.7 × 103 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 (Kapp = 2.8 × 102 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.
Circular dichroism, Secondary structure, Ligand binding, Materials, Biochemistry