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. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane. However, the interaction between presequence peptides and Tim23p remains unclear. Herein, we investigated the interaction of presequence peptides with the intermembrane space domain of Tim23p (Tim23(IMS)) by fluorescence and micro-Raman spectroscopy. The fluorescence quenching revealed that the interaction between Tim231(IMS) and presequence peptides is mainly electrostatic interaction. Micro-Raman spectroscopy and ANS binding experiments showed that presequence peptides induce a more compact conformation of Tim231(IMS). GST pull-down experiments and tryptophan fluorescence indicated that there is no interaction between Tim231(IMS) and Tim50(IMS). (C) 2013 Elsevier Inc. All rights reserved.