High-resolution NMR spectroscopy of proteins in intact mitochondria

Understanding protein structure and function within mitochondria is essential for unraveling the molecular mechanisms underlying cellular energy production, stress response and disease. Here we present an approach for NMR observation of proteins within intact mitochondria by delivering proteins directly into isolated mitochondria via electroporation. Using this method, we investigate the interaction of α-synuclein with the mitochondrial membrane and examine how post-translational modifications regulate this interaction. In addition, we assessed the stability of GB1 and the dimerization of its variant within mitochondria, achieving quantitative insights into mitochondrial environmental impact on protein function. This approach offers a valuable framework for exploring mitochondria-related biomolecular events at atomic resolution within intact mitochondria, paving the way for a more comprehensive understanding of the molecular events governing mitochondrial health and dysfunction.