title: Expression and structural characterization of human translocase of inner membrane of mitochondria Tim50.
abstract: The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor in TIM23 complex, which spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. In this study, we expressed and purified the intermembrane space (IMS) domain of human Tim50 (Tim50(IMS)), and investigated its structural characteristics and assembly behaviors. The far-UV CD spectra of Tim50(IMS) in native and denatured states revealed that the protein has a significantly folded secondary structure consisted of α-helixes and β-sheets. Size exclusion chromatography showed that Tim50(IMS) is a monomer. Furthermore, the results showed, by intrinsic fluorescence, ANS binding, fluorescence anisotropy and fluorescence quenching, that Tim50(IMS) forms a compact structure in the range of pH 8.0-5.0; and it is more compact at pH 8.0 than pH 7.0; when pH decreases below 5.0, the protein is gradually denatured.
authors: The Key Laboratory of Bioactive Materials, Ministry of Education, School of Physics Science, Nankai University, Tianjin 300071, PR China.
authors: Yongqiang
authors: Y
authors: Zhang
authors: Yun
authors: Y
authors: Xu
authors: Qing
authors: Q
authors: Zhao
authors: Zhina
authors: Z
authors: Ji
authors: Qiang
authors: Q
authors: Li
authors: Shu Jie
authors: SJ
authors: Li
mesh_terms: D002460:Cell Line; D002942:Circular Dichroism; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D051336:Mitochondrial Membranes / Q000737:chemistry*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C509348:TIMM50 protein, human