The structural characteristics of human preprotein translocase of the inner mitochondrial membrane Tim23: Implications for its physiological activities

详细信息    查看全文

• 作者：Yongqiang Zhang^a ; ^b ; ¹ ; Yun Xu^a ; ^c ; ¹ ; Qing Zhao^a ; Zhina Ji^a ; Honghua Deng^a ; Shu Jie Li^a ; ^{shujieli@nankai.edu.cn}
• 关键词：Mitochondria ; TIM23 complex ; Tim23 ; Conformation ; Dimer
• 刊名：Protein Expression and Purification
• 出版年：2012
• 出版时间：April, 2012
• 年：2012
• 卷：82
• 期：2
• 页码：255-262
• 全文大小：661 K

文摘

The preprotein translocase of the inner mitochondrial membrane (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23 forms a pore for preprotein transportation in TIM23 complex, which spans the inner membrane with transmembrane segments and exposes a hydrophilic domain in the intermembrane space. In this study, we expressed and purified the intermembrane space (IMS) domain of human Tim23 (Tim23_IMS). The far-UV CD spectra of Tim23_IMS in native and denatured states revealed that the protein has a limited secondary structure and a not well-defined tertiary packing. Its Stokes radius was larger than both its expected size as a folded globular protein and the size determined by size exclusion chromatography. A large increase in 8-anilino-1-naphthalene-sulfonate (ANS) fluorescence (>50-fold) was observed, indicating that hydrophobic clusters are exposed at its surface. And GlobPlot/DisEMBL program predicted that the protein is in a loose folding state. We therefore conclude that, the non-bound hydrophilic domain of the human Tim23 is in a molten globule configuration with marginal stability. Furthermore, size exclusion chromatography and sedimentation equilibrium analysis showed that Tim23_IMS exists as a dimer. And the results, showed by ANS binding and fluorescence quenching, indicated that a pH-dependent conformational change of Tim23_IMS occurs, and at pH 4 and 3, it forms a compact structure.