Extended Data Fig. 8: TMEM65 fluorescence measurements.

a, Size-exclusion chromatography of purified TMEM65. TMEM65 eluted at 11.9 mL on a Superdex 200 column. Image: SDS–PAGE and Coomassie staining of the peak fraction. b-d, Fluorescence changes of WT (red) and D132A (black) TMEM65 upon addition of cations. Panels b and d display Trp fluorescence changes with 20 mM Na⁺ and 20 µM Ca²⁺, respectively, while panel c shows Tyr fluorescence changes with 20 mM Na⁺. Notably, Na⁺ and Ca²⁺ induce opposite changes in Trp fluorescence, suggesting that these cations drive the conformational distribution of TMEM65 towards two distinct conformations, possibly matrix- versus IMS-facing conformations. We used 30% more D132A TMEM65 (13 µg per experiment) than WT (10 µg) to create a more stringent test of our hypothesis that D132A would diminish TMEM65 fluorescence changes. Despite this, WT TMEM65 still exhibits significantly larger fluorescence changes upon cation addition. Each data point represents results from an independent protein preparation. Statistical analysis was performed using unpaired, two-tailed t-test. We note that a titration experiment to determine Na⁺ or Ca²⁺ affinity of TMEM65—while impractical in the current study due to the modest fluorescence changes observed even at high Na⁺ or Ca²⁺ concentrations—may be feasible in future studies in which Trp residues are engineered to positions expected to undergo more substantial changes in their chemical environments upon ion binding. All data in this figure are presented as mean ± SEM. Statistical analysis was performed using unpaired, two-tailed t-test. All experiments were performed with 3 independent biological replicates.

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