To develop an NMR spectroscopy method for examining protein structure, interactions, and stability inside intact mitochondria.
Approach:
Mitochondrial preparation: Researchers isolated mitochondria from rat liver and pig brain, then used electroporation to deliver isotope-labeled proteins produced in Escherichia coli.
Validation and measurements: Membrane-potential and enzyme-activity tests assessed mitochondrial integrity and function. Nitrogen-15 and fluorine-19 NMR were used to study alpha-synuclein, GB1, ubiquitin, and KH1.
Key Findings:
Most measured signals came from proteins inside mitochondria, and tests indicated the organelles remained intact and functional after electroporation.
Alpha-synuclein remained largely disordered, while its N-terminal region interacted with mitochondrial membranes.
The Parkinson’s-associated A30P variant and methionine oxidation each reduced alpha-synuclein’s membrane interaction.
GB1 was more stable inside mitochondria than in buffer, and binding between two copies of a GB1 variant was approximately twice as strong inside mitochondria.
Interpretation:
The results show that the mitochondrial environment can affect protein stability and interactions. The study did not assess disease progression or patient outcomes.
Limitations:
Experiments used isolated mitochondria at temperatures below normal physiological conditions.
Samples remained viable for a limited period, restricting study of slower processes.
Further work is needed to improve sensitivity and sample viability and to examine additional proteins and disease models.
Conclusion:
The method enables NMR study of selected proteins within intact mitochondria.
Optical analysis of resection-margin tissue identified tumor infiltration as the strongest contributor to predictions of where glioblastoma later recurred.
