Expression and structural characterization of human translocase of inner membrane of mitochondria Tim50

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 (Tim50IMS), and investigated its structural characteristics and assembly behaviors. The far-UV CD spectra of Tim50IMS 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 Tim50IMS is a monomer. Furthermore, the results showed, by intrinsic fluorescence, ANS binding, fluorescence anisotropy and fluorescence quenching, that Tim50IMS 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.

► Tim50 is a major receptor in TIM23 complex.
► We determined its structural characteristics, assembly behaviors and pH-dependent conformational changes.
► Tim50 has a significantly folded secondary structure consisted of α-helixes and β-sheets.
► Tim50 forms a monomer.
► Tim50 is more compact at pH 8.0 than pH 7.0 and unfolded at pH 3.0.