Scientists Watch Proteins at Work Inside Intact Mitochondria Using NMR

Mitochondria have long been described as the powerhouses of the cell, but for structural biologists they have remained frustratingly opaque. These organelles host some of the most consequential chemistry in human biology, from oxidative phosphorylation to the stress responses that decide whether a cell lives or dies, yet the behavior of individual proteins inside them has been largely invisible at the atomic level. Now a team at the Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences reports in Nature Chemical Biology a method that changes this picture. By combining electroporation with high-resolution nuclear magnetic resonance (NMR) spectroscopy, the researchers have for the first time obtained residue-resolved spectra of proteins functioning inside intact, isolated mitochondria, opening a new window onto the molecular events that govern mitochondrial health and dysfunction.

The central obstacle to studying proteins inside mitochondria has always been delivery combined with signal quality. In-cell NMR, a technique pioneered nearly two decades ago, allows researchers to observe isotopically labeled proteins inside living cells, but applying the same logic to a subcellular organelle is far harder. Mitochondria are small, bounded by a double membrane, and easily damaged during isolation. Earlier work, including a 2014 study by Barbieri, Luchinat and Banci, demonstrated that in-mitochondria NMR was conceptually possible, but the approach has remained technically demanding and limited in resolution. The new study, led by Conggang Li with co-first authors Zeting Zhang and Cai Zhang, builds on that foundation and pushes the method to the point where quantitative, atomic-resolution measurements become routine enough to answer biological questions.

The key innovation lies in sample preparation. The team delivers purified proteins directly into isolated mitochondria using electroporation, a technique in which brief electrical pulses create transient pores in membranes, allowing macromolecules to enter. Crucially, the researchers performed extensive control experiments to verify that the delivered proteins genuinely resided inside the organelles rather than clinging to the outer membrane or leaking into the surrounding buffer. By comparing NMR spectra of the mitochondrial samples with spectra of the supernatant after centrifugation, they confirmed that the signals they recorded arose from proteins within intact mitochondria. They also quantified the intramitochondrial concentration of each delivered protein by lysing the organelles after measurement and comparing integrated peak areas against an internal standard, a step that transforms the method from a qualitative observation into a genuinely quantitative tool.

Two complementary labeling strategies underpin the spectroscopy. For residue-resolved information, the team used uniformly nitrogen-15-enriched proteins and recorded two-dimensional proton-nitrogen correlation spectra, the workhorse experiment of solution NMR. Each cross-peak in such a spectrum corresponds to a specific atom pair in a specific amino acid residue, so changes in peak position, intensity or width report directly on what is happening to that part of the protein. In parallel, the researchers exploited fluorine-19 labeling, incorporating fluorinated amino acid analogues such as 3-fluorotyrosine and 5-fluorotryptophan into their target proteins. Fluorine-19 offers a background-free window into biological samples, because essentially no endogenous mitochondrial molecules contain fluorine, and modern fluorine probes, including the highly sensitive wPSP6F tag developed in the same laboratory, boost sensitivity further. Together, the two approaches allow the same molecular event to be viewed from complementary angles.

The biological centerpiece of the study is alpha-synuclein, the intrinsically disordered protein whose aggregation into Lewy bodies is a hallmark of Parkinson’s disease. Alpha-synuclein has long been suspected of interacting with mitochondrial membranes, particularly cardiolipin-rich regions of the inner membrane, and this interaction is thought to contribute to the mitochondrial dysfunction seen in the disease. Using their in-mitochondria NMR platform, the researchers observed how alpha-synuclein engages the mitochondrial membrane environment, with residue-resolved signal attenuation revealing which parts of the protein chain make contact with membranes. The spectra of nitrogen-15-enriched alpha-synuclein delivered into mitochondria isolated from rat liver and pig brain showed characteristic broadening and attenuation patterns that map the membrane-binding interface at single-residue resolution, something previously achievable only with simplified model membranes in a test tube.

Perhaps the most striking aspect of the work is its treatment of post-translational modifications, the chemical tags that cells attach to proteins to regulate their behavior. The team examined how modifications of alpha-synuclein, including phosphorylation and methionine oxidation, alter its interaction with the mitochondrial membrane. Oxidation is of particular interest because mitochondrial metabolism generates reactive oxygen species, and oxidized alpha-synuclein has been implicated in impaired clearance and altered chaperone binding. The researchers also probed how oxidized alpha-synuclein interacts with the mitochondrial chaperone TRAP1 and with protein disulfide isomerase, using methionine-selective nitrogen-15 enrichment to monitor the oxidation state directly. These experiments demonstrate that the platform can capture regulatory chemistry as it happens inside the organelle, not merely static structure.

To establish the generality of the method, the team turned to a well-characterized benchmark: the B1 domain of protein G, known as GB1, a small, stably folded globular protein that has served as a standard in NMR methodology development for years. By measuring GB1 inside mitochondria, the researchers could assess how the mitochondrial interior, with its crowded macromolecular environment, distinctive pH and high ionic strength, affects protein stability. They also engineered a GB1 variant and monitored its dimerization inside the organelle using fluorine-19 NMR, achieving quantitative insight into how the mitochondrial environment modulates protein-protein interactions. This matters because a growing body of evidence shows that the intracellular milieu, with its excluded-volume effects and specific weak interactions, can shift protein folding equilibria and binding affinities in ways that dilute buffer experiments cannot reproduce.

The technical rigor behind these measurements deserves emphasis. The team measured the internal pH of their mitochondrial preparations using the fluorescent dye BCECF and cross-checked it against NMR chemical shifts of alpha-synuclein, ensuring that spectral changes reflected genuine intramitochondrial conditions rather than artifacts of sample preparation. Deuterated GB1 was used to probe hydrogen exchange and protein dynamics inside the organelle, with time-resolved spectra showing how signals recover as amide protons exchange with solvent. Leakage controls, concentration quantification and comparisons between buffer and mitochondrial samples were performed for every protein studied, establishing a level of validation that will be essential if the method is to be adopted by other laboratories.

The implications extend well beyond method development. Mitochondrial dysfunction sits at the heart of Parkinson’s disease, Alzheimer’s disease, cardiomyopathy, diabetes and aging itself, yet most structural biology on mitochondrial proteins has been performed on purified components or membrane mimetics. A platform that reports on protein structure, stability and interactions inside intact mitochondria bridges the gap between reductionist test-tube biophysics and the messy reality of the organelle. It also complements other emerging approaches, such as dynamic nuclear polarization-supported solid-state NMR, which has recently been used to study nucleic acids and proteins in situ with subcellular specificity, and real-time NMR observations of endogenous cytochrome c translocation within mitochondria reported in 2024. Together these techniques sketch a future in which the atomic-resolution study of biology inside organelles becomes standard practice rather than a tour de force.

For the immediate future, the Wuhan team’s framework offers a template for exploring mitochondria-related biomolecular events that were previously out of reach: how disease-linked mutations change membrane binding, how chaperones protect proteins in the crowded mitochondrial interior, and how the organelle’s unique chemical environment reshapes the energy landscape of folding and aggregation. As the authors note, the approach paves the way toward a more comprehensive understanding of the molecular events governing mitochondrial health and dysfunction. If the method proves as adaptable as in-cell NMR in whole cells has been, the coming years could see atomic-resolution spectroscopy become a routine probe of the organelle whose failure underlies so much of human disease, finally letting researchers watch, atom by atom, what goes wrong inside the powerhouse.

Subject of Research: High-resolution NMR spectroscopy of proteins delivered into intact mitochondria

Article Title: High-resolution NMR spectroscopy of proteins in intact mitochondria

Article References: Zhang, Z., Zhang, C., Xu, G., Du, R., Yu, J., Liu, X., Chai, Z., Wu, Q., Jiang, L., Liu, M., & Li, C. (2026). High-resolution NMR spectroscopy of proteins in intact mitochondria. Nature Chemical Biology. https://doi.org/10.1038/s41589-026-02314-x

Image Credits: AI Generated

DOI: 10.1038/s41589-026-02314-x

Keywords: NMR spectroscopy, mitochondria, in-cell NMR, alpha-synuclein, Parkinson's disease, electroporation, fluorine-19 labeling, protein stability, post-translational modifications, mitochondrial membrane, structural biology, protein dimerization