Crystallization and Interaction of Histidine-Containing Tetrapeptides with Metal Ions

Recent advancements in the study of supramolecular structures and crystals of peptides have been particularly significant at the intersection of nanotechnology and biotechnology. Diphenylalanine, composed of two phenylalanine residues, is known for its multiple self-assembling structures and functional properties, such as luminescence and piezoelectricity[1]. Building on this foundation, tetrapeptides inspired by diphenylalanine have demonstrated the ability to form dense hydrogen bond networks in crystals, promising the design of more functional peptide crystals with desirable properties[2].<br/><br/>Research has also been active in stabilizing structures and imparting new functions by adding metal ions to short peptides. For instance, metal ions are known to facilitate the fibrillization of amyloid peptides[3]. Additionally, artificial amyloid peptides containing histidine have formed nanowires with metal ions, exhibiting catalytic activity[4]. The imidazolium ring in histidine's side chain can coordinate with various metal ions, suggesting that peptides containing histidine could facilitate and functionalize peptide crystals if the design rules become clearer.<br/><br/>This study aims to design a new tetrapeptide containing histidine and examine its co-crystallization conditions with metal ions. To elucidate the intermolecular interactions between peptide molecules and metal ions, we utilized polarized Raman spectroscopy to investigate the peak shifts and anisotropy of vibrational modes in amino acids.<br/><br/>A tetrapeptide, HIIH, with histidine (H) at both termini was designed. Isoleucine (I), known for its hydrophobicity and promotion of β-sheet formation, was included to stabilize the crystal structure through hydrophobic interactions. Copper chloride was selected as the metal salt due to the well-documented interactions between copper ions and histidine in nature. Crystallization was attempted for the peptide alone and with a 1:1 molar ratio of peptide to metal ions using the vapor diffusion method, commonly employed in protein crystallization. Crystals were observed using optical and polarized light microscopy, and changes in chemical bonding within the crystals were investigated using polarized micro-Raman spectroscopy.<br/><br/>Crystals were obtained for both HIIH alone and HIIH mixed with copper ions. Both formed needle-like transparent crystals, with colorless crystals for the peptide alone and green crystals when mixed with copper ions. Raman spectra between 1550 cm^-1 and 1700 cm^-1, corresponding mainly to stretching modes of double bonds, showed significant changes when copper chloride was mixed with HIIH. The 1650-1700 cm^-1 region, known as the Amide I band, corresponds to the C=O stretching mode in peptide amide bonds[5]. The addition of copper ions resulted in a shift to lower wavenumbers without significant changes in polarization dependency, suggesting a shortening of intermolecular distances without altering the structure. In contrast, multiple peaks in the 1550-1650 cm^-1 range, corresponding to double bonds in the imidazolium ring, changed both in peak positions and polarization dependence[6]. This signature indicated that copper ions selectively influence the imidazolium rings in the peptide crystal. These findings provide insights into establishing design rules for tetrapeptides integrating with ions for peptide crystallization.<br/><br/>[1] Tao, Kai, et al., Science 358.6365 (2017): eaam9756.<br/>[2] Motai, Kazunori, et al., Crystal Growth & Design 23.6 (2023): 4556-4561.<br/>[3] Makhlynets, et al., Angewandte Chemie International Edition 55.31 (2016): 9017-9020.<br/>[4] Rufo, Caroline M., et al., Nature chemistry 6.4 (2014): 303-309.<br/>[5] Hiroo Hamaguchi, Raman spectroscopy, Kodansha (2015): 110.<br/>[6] Hideo Takeuchi, Bunkoukenkyu 59.2 (2010): 73.