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- Application of the SAIL method to the studies of protein-ligand interactions
Chun-Jiun Yang, Graduate School of Science and Engineering, Department of Chemistry
| Over the last several decades, there have been enormous advances in the field of biomolecular NMR (Nuclear Magnetic Resonance), including the advents of highly sensitive hardware, effective isotope-labelling methods, new NMR experiments, and automatic analyses of NMR signals. However, there are still some situations in which NMR has not been optimized for protein studies. The SAIL (Stereo-Array Isotope Labelling) method, developed by Prof. Kainosho and his colleagues, provides unique opportunities for studying proteins by NMR [1]. In addition to the well-known application for the structure determination of large-size proteins at atomic resolution, the SAIL method is applicable for studying protein dynamics. |
| Here, we have extended the SAIL method to the observation of protein-ligand interactions, as this will accelerate the analysis of many biological processes such as a cell surface receptor-ligand interaction and thereby serve for the development of molecular-targeted drugs. In particular, we use aromatic residues (i.e. His, Phe, Trp, and Tyr) as probes to detect the interactions. Aromatic residues play important roles in hydrophobic core formation in many proteins. The residues are frequently found in the interfaces of protein-protein and protein-ligand complexes, where they contribute to hydrophobic contacts. However, NMR studies of their detailed roles are lagging behind due to technical difficulties, such as the narrow dispersion of the aromatic NMR signals and the strong and complicated couplings between the aromatic atoms in the conventionally isotope-labelled protein samples. |
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In this project, we have overcome these difficulties by introducing various types of SAIL-Phe and -Tyr amino acids into a protein. The SAIL-Phe and -Tyr enable precise observations of the changes of NMR signals upon ligand binding. In addition, the ligand-induced changes of the dynamics in the aromatic region are detectable. We chose the FKBP-12 and FKBP-12.6 proteins as model systems (Fig. 1).
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Fig. 1. Structures of FKBP-12 (left) and FKBP-12.6 (right). |
| Both FKBP-12 and FKBP-12.6 belong to the FKBP family (FK506 binding protein). FKBP functions as a peptidyl-prolyl cis-trans isomerase and has been found in many eukaryotic cellular signaling pathways, from yeast to human. FKBP binds to rapamycin as well as FK506, both of which are potent immunosuppressants frequently used for organ transplantation, by hydrophobic interactions via aromatic residues. With mediation by rapamycin and FK506, FKBP-12 can form complexes with mTOR (mammalian target of rapamycin) and calcineurin, respectively, thereby inhibiting their functions. FKBP-12 and FKBP-12.6 share 85% sequence identity with each other. Among its 107 residues, FKBP-12 contains 3 Tyr and 5 Phe residues, while FKBP-12.6 has 2 Tyr and 6 Phe. |
| We selectively labelled both proteins with SAIL-Phe or –Tyr, and subsequently observed the signal changes upon ligand binding. For instance, Fig. 2 shows the 2D [1H, 13C] HSQC spectra of FKBP-12.6, labelled by ζ-SAIL Phe, without and with ligands.
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Fig. 2. Overlaid 2D [1H, 13C] HSQC spectra of FKBP-12.6 labelled with ζ-SAIL Phe.
The different colours represent the conditions without and with ligands.
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| The clarity of the data convincingly demonstrates the usefulness of the SAIL method for the observation of protein-ligand interactions. We are now expanding the study to elucidate the underlying dynamics. |
[1] Kainosho, M., Torizawa, T., Iwashita, Y., Terauchi, T., Mei Ono, A., and Güntert,
P. (2006) Nature 440, 52-57
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