4.10. Metal Ion Binding Assay

The metal ion binding capacity of VVA2 variants was conducted based on intrinsic tryptophan fluorescence (ITF) measurements [40,41], as previously reported. Recombinant VVA2 variants without free metal ions (pre-treated with EDTA and buffer exchange, EDTA: Ethylenediaminetetraacetic acid) were diluted to 10 µM in assay buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl). Metal ions (Mg²⁺ or Mn²⁺) were added to the final concentration of 20 mM. A Tecan Spark 10M microplate reader was used to measure the intrinsic fluorescence. Excitation was set as 290 nm, while emission scan was set as 310–440 nm. The bandwidth was adjusted to 5 nm, with a step size of 2 nm. Background fluorescence was deducted using the buffer with a similar operation. Spectra with more than three repeats were analyzed and presented with GraphPad Prism.