The structural characteristics of human preprotein translocase of the inner mitochondrial membrane Tim23: implications for its physiological activities.
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.
The Key Laboratory of Bioactive Materials, Ministry of Education, School of Physics Science, Nankai University, Tianjin 300071, PR China.
Yongqiang
Y
Zhang
Yun
Y
Xu
Qing
Q
Zhao
Zhina
Z
Ji
Honghua
H
Deng
Shu Jie
SJ
Li
D002850:Chromatography, Gel; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013499:Surface Properties; D014508:Urea / Q000737:chemistry
D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C438387:TIMM23 protein, human; D014508:Urea