Supplementary Figure 1: Crystallized chimera construct (mHv1cc)
From: X-ray crystal structure of voltage-gated proton channel
(a) Sequence alignment between mHv1cc and other VSDs. These sequences (mHv1cc, Kv1.2–Kv2.1; shaker family voltage–gated potassium channel Kv1.2–Kv2.1 paddle chimera channel, NavAb; voltage–gated sodium channel from Acrobacter butzleri, Kv1.2; voltage–gated potassium channel subunit Kv1.2, KvAP; voltage–gated potassium channel from Aeropyrum pemix, NavRh; NaChBac orthologue from the marine alphaproteobacterium HIB114, and Ci–VSP; voltage sensor phosphatase from Ciona intestinalis were aligned using program Clustal W39. The red characters show the periodically basic residues on S4. The green characters show the hydrophobic residues highly conserved among VSDs. (b) The cartoon diagram of crystallized chimera constructs (mHv1cc). The S2–S3 half intracellular–side (Glu149–Phe171) and cytoplasmic coiled–coil (Val216–Asn269) of mouse Hv1 or VSOP were replaced with the intracellular portion of Ci–VSP (Asp164 – Leu188) and the leucine zipper transcriptional activator GCN4 from S. cereviece (Arg249–Arg281), respectively. The intracellular portion of Ci–VSP and GCN4 show red and green diagrams, respectively. (c, d) Thermal stability assay of mHv1cc with CPM. (c) A raw fluorescence signal plotted against increasing temperature. (d) Melting temperature (Tm) was determined as the first inflection point by calculating derivative of raw fluorescence shown in a against temperature. Tm of mHv1cc was estimated to be 70.6 °C. The red and blue show plots with (n = 4 technical replicates) and without mHv1cc (n = 2 technical replicates), respectively. (e, f) Proton current of mHv1cc recorded at the indicated two different pH environments from HEK293T cells. (g) Stoichiometry of mHv1cc in HEK293T cells as shown by cross–linking with DSS. The arrow shows a band corresponding to dimer of mHv1cc. (h) H+ flux assay using proteoliposome. Data from vesicles with and without mHv1cc were shown in red and black curves, respectively. Error bars depict means ± S.D. (n = 3 technical replicates).
