Miller S, Schuler B, Seckler R
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Biochemistry. 1998 Jun 23;37(25):9160-8. doi: 10.1021/bi980190e.
The homotrimeric tailspike endorhamnosidase of phage P22 has been used to compare in vivo and in vitro folding pathways and the influence of single amino acid substitutions thereon. Its main structural motif, which contains the known folding mutation sites, consists of three large right-handed parallel beta-helices. A thermodynamic analysis of the stability of tailspike is prevented by the irreversibility of unfolding at high temperatures or high concentrations of denaturant, probably due to interdigitation of the domains neighboring the beta-helix. We therefore expressed and isolated a tailspike fragment comprising only its central beta-helix domain (residues 109-544). As shown by equilibrium ultracentrifugation, the isolated beta-helix is a monomer at concentrations below 1 microM and trimerizes reversibly at higher protein concentrations. Both the similarity of fluorescence and CD spectra, compared to the complete protein, and the specific binding and hydrolysis of substrate suggest a nativelike structure. Moreover, urea denaturation transitions of the beta-helix domain are freely reversible, providing the basis for a future quantitative analysis of the effects of the folding mutations on the thermodynamic stability of the domain and of structural features responsible for folding and stability of the parallel beta-helix motif in general.
噬菌体P22的同源三聚体尾刺内鼠李糖苷酶已被用于比较体内和体外折叠途径以及单个氨基酸取代对其的影响。其主要结构基序包含已知的折叠突变位点，由三个大型右手平行β-螺旋组成。由于在高温或高浓度变性剂下展开的不可逆性，可能是由于与β-螺旋相邻的结构域相互交错，阻碍了对尾刺稳定性的热力学分析。因此，我们表达并分离了一个仅包含其中心β-螺旋结构域（第109-544位氨基酸残基）的尾刺片段。平衡超速离心表明，分离出的β-螺旋在浓度低于1 microM时为单体，在较高蛋白质浓度下可逆三聚化。与完整蛋白质相比，荧光和圆二色光谱的相似性以及底物的特异性结合和水解表明其具有类似天然的结构。此外，β-螺旋结构域的尿素变性转变是完全可逆的，为未来定量分析折叠突变对该结构域热力学稳定性的影响以及一般负责平行β-螺旋基序折叠和稳定性的结构特征奠定了基础。
