[unreadable] [unreadable] Nearly all mitochondrial proteins are encoded by the nuclear genome and imported post-translationally into the organelle. The TIM23 complex mediates the translocation of precursor proteins across the inner membrane (IM) of mitochondria, as well as protein integration into the IM; however, many structural and mechanistic aspects of these processes are poorly understood. In the following proposed research, fluorescent and photoreactive probes will be incorporated into specific sites in nascent chains of mitochondrial integration intermediates and full-length functional translocon components. Using fluorescence spectroscopy and photocrosslinking techniques, a unique experimental approach will be used to address several fundamental issues regarding TIM23-mediated membrane protein integration. By directly monitoring the molecular environment, the compartmental location, and adjacent proteins of specific substrate regions during defined stages of integration, this work will yield a high-resolution analysis of all stages of IM protein topogenesis. Similarly, by positioning probes within specific regions of the TIM23 translocon itself, this work will provide key information regarding translocon dynamics and protein interactions associated with channel formation and substrate import, as well as the nature and identity of the gating mechanism. In addition to providing mechanistic and structural insights into the integration process, this novel method of studying mitochondrial import has strong potential for practical advances in mitochondrial pathogenic research. [unreadable] [unreadable] [unreadable]