Selection of a platinum-binding sequence in a loop of a four-helix bundle protein

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Protein–metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications.

Section snippets

Construction of the phage display LARFH library

To create an LARFH gene library, amino acid residues at positions 21, 22, 23, 25, and 26 were subjected to semi-random mutagenesis (Fig. 1). Each of their codons was replaced with the NVS codon (N = A + T + G + C, V = A + G + C, S = G + C). Genes were then amplified by using two different primers so that the EcoRI and HindIII recognition sites could be added at the 5′ and 3′ termini, respectively. Amplified DNA fragments were digested with EcoRI and HindIII and then ligated to T7 Select Vector

Selection of mutant LARFH that interacts with platinum

Fig. 1A shows the amino acid sequence of LARFH, with rectangles representing segments that are likely to form α-helices. We inserted a platinum-binding sequence in the loop connecting the first and second α-helices. When LARFH is displayed on phage particles, the first loop may be positioned at the opposite face of the phage and may be well exposed to solvent (Fig. 1B). Therefore, we considered that platinum and other metal particles might access this loop more easily. The residues Ser21,

Discussion

In this study, we successfully inserted a high-affinity platinum binding site in LARFH, which enabled the protein to site-specifically bind to a platinum surface. The mutant protein with an YKRGYK sequence in its first loop seems to bind to platinum more strongly than wild-type LARFH. The KD of mutant LARFH binding to platinum is 1.0 × 10−8 M, which is better than those of peptides whose binding constants have been previously measured (21); for example, 3l-PtBP2 has a reported Keq of 6.7 × 106 M

Acknowledgments

The work was supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S1512002), 2015–2017 and by JSPS KAKENHI Grant Number 16K14494.

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