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<p>Mitochondrial outer membrane proteins (β-OMPs) serve as the first line of communication with the cellular milieu. A crucial β-OMP, the sorting and assembly machinery Sam50, is a 16-stranded transmembrane β-barrel highly conserved in all eukaryotes. Sam50 dysregulation is lethal; yet, molecular elements that regulate Sam50 remain poorly understood. Here, we identify and characterize residues that regulate Sam50 structure and function. Using single-molecule electrophysiology, in vivo function, and stability measurements, we demonstrate that the POlypeptide-TRansport Associated domain is dispensable for Sam50. Complete characterization of the folding mechanism using 165 Xaa→Ala substitutions reveals that Sam50 folds through parallel pathways, with at least two transition states. The folding nucleus is towards the N-terminus, whereas frustrated folding at the C-terminal region kinetically traps the structure. We correlate this unexpected folding of Sam50 with its SAM-assisted assembly. Additionally, our per-residue stability measurements show that destabilizing hotspots in Sam50 are linked to its gating function. Our findings suggest how the dynamic structure of Sam50 offers a functional advantage, with specific residues that regulate folding, stability, and function, also determine the protein’s sensitivity to mutations.</p>

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Linking multipoint folding and stability with functional regulation in the mitochondrial transmembrane β-barrel Sam50

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月2日

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  • Roshika Ravi,
  • Swadha Gupta,
  • Jyoti Kumari,
  • Aadish Rawat,
  • Radhakrishnan Mahalakshmi

摘要

Mitochondrial outer membrane proteins (β-OMPs) serve as the first line of communication with the cellular milieu. A crucial β-OMP, the sorting and assembly machinery Sam50, is a 16-stranded transmembrane β-barrel highly conserved in all eukaryotes. Sam50 dysregulation is lethal; yet, molecular elements that regulate Sam50 remain poorly understood. Here, we identify and characterize residues that regulate Sam50 structure and function. Using single-molecule electrophysiology, in vivo function, and stability measurements, we demonstrate that the POlypeptide-TRansport Associated domain is dispensable for Sam50. Complete characterization of the folding mechanism using 165 Xaa→Ala substitutions reveals that Sam50 folds through parallel pathways, with at least two transition states. The folding nucleus is towards the N-terminus, whereas frustrated folding at the C-terminal region kinetically traps the structure. We correlate this unexpected folding of Sam50 with its SAM-assisted assembly. Additionally, our per-residue stability measurements show that destabilizing hotspots in Sam50 are linked to its gating function. Our findings suggest how the dynamic structure of Sam50 offers a functional advantage, with specific residues that regulate folding, stability, and function, also determine the protein’s sensitivity to mutations.