Nakamura Chikashi, Takeda Seiji, Kageshima Masami, Ito Miyuki, Sugimoto Naoki, Sekizawa Kazuko, Miyake Jun
Tissue Engineering Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan.
Biopolymers. 2004;76(1):48-54. doi: 10.1002/bip.10572.
Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.
此前有报道称，一些肽可与低分子量化学物质结合。从噬菌体文库中筛选出一种氨基酸序列为His-Ala-Ser-Tyr-Ser的肽，它能与阳离子卟啉5,10,15,20-四（N-甲基吡啶-4-基）-21H,23H-卟吩（TMpyP）选择性结合，结合常数为10⁵ M⁻¹（J. Kawakami、T. Kitano和N. Sugimoto，《化学通讯》，1999年，第1765 - 1766页）。推测这种结合是由于组氨酸和酪氨酸这两种芳香族氨基酸的π电子堆积所致。在本研究中，利用原子力显微镜（AFM）通过力曲线分析进一步研究了TMpyP与该肽之间的弱相互作用。通过AFM针尖的垂直移动来测量解开肽-卟啉复合物所需的机械力。在涂金云母基底和涂金AFM针尖上均形成了肽自组装单分子层。当固定有肽的AFM针尖在含有TMpyP的溶液中与固定有肽的基底接触时，TMpyP可结合在两层肽之间。在缩回过程中，观察到一种使TMpyP与肽之间的相互作用断裂的力。解离力值与TMpyP的浓度相关。力测量得到的卟啉检测限为100 ng/mL，与表面等离子体共振传感器的检测限相似。此外，我们计算了观察到的力与分子伸长长度的乘积，以确定解开复合物所需的功。与卟啉-肽的结合能相比，得到的解离功值在合理范围内。
