Mn,Zn-MTGFP融合蛋白磁性與結構分析

Abstract

金屬硫蛋白與綠色螢光蛋白所形成的融合蛋白（MT-GFP）是一個內含金屬鍵結的蛋白質，此蛋白質含有20個高度保留的半胱胺酸(cysteine,Cys)容易與重金屬離子形成硫金屬鍵結。其中含有七個二價過渡性金屬離子和兩個類似Zinc-Blende金屬鍵結基團。在這項研究中，我們將Zn2+以及 Mn2+ 取代MT-GFP 原有的結合金屬形成錳鋅螢光金屬硫蛋白，並且發現錳鋅螢光金屬硫蛋白經由超導量子干涉儀測量，在10K到300K的範圍中展現出弱鐵磁性的特性。再經由拉曼光譜儀的檢測，可以發現鋅離子和錳離子係藉由與金屬硫鍵結結合在蛋白質中，分別在288 cm-1和355 cm-1有Zn-S以及Mn-S之Bending mode拉曼光譜訊號。經由延伸X射線吸收精細結構（EXAFS光譜）的分析更加確定Mn2 +經由硫與MT-GFP結合。Mn,Zn-MT-GFP更可以用TEM的影像圖以及電子繞射圖形推測其結構。

Metallothionein-green fluorescence fusion protein (MT-GFP) is a zinc binding protein, which binds seven divalent transition metal ions through its 20 conserved cysteines and forms two metal binding clusters with Zinc-Blende structure. In this study, Mn2+ ions has substituted for Zn2+ at M3, M4 metal binding sites in the β-domain of MT-GFP. We found this Mn, Zn binding protein exhibits weak ferromagnetic properties at temperature range from 10K to 300K by SQUID measurement. By micro-Raman spectroscopy analysis, the Zn-S and Mn-S bending modes can be observed clearly at 288 cm-1 and 355 cm-1, respectively. These evidences indicate that the Zn2+ and Mn2+ ions are bound with Cys residues of MT-GFP. Extended X-ray absorption fine structure (EXAFS) analysis also indicats Mn2+ ions bond to MT-GFP via the Mn-S bond of Cys. The conformation of Mn,Zn-MT-GFP can be characterized by TEM and the its intermolecular interactions can be determined by its electron diffraction pattern.