The metal occupancy of cytosolic metal-binding proteins is predicted from the thermodynamic activities of metals in a cell.
These activities (as standard free-energies, ΔG°) have been determined in a bacterial cell (Osman et al. (2019) Nat. Chem. Bio. 15: 241–249).
The calculator is based on Young et al (2021) Nature Communications 1195
The resulting predictions can:
- Identify metal-specificity
- Infer mis-metalation
- Reveal erroneous affinity measurements
- Suggest differences between these thermodynamic metal activities and those of the host cell
- Indicate where additional mechanisms (such as molecular interactions) must assist metalation
You can access the beta-versions of a metalation calculator here
Notes about kinetics: (1) Cytosolic available metal is typically bound (and buffered). However, the buffer can become depleted if the rate of demand, for example for an over-expressed recombinant protein, greatly exceeds the rate of metal-import. Metal availability then drops below the steady-state [available metal] (really an activity) value in the calculator. DNA-binding metal sensors, tuned to such changes, have been used to read-out bespoke values enabling speciation of metalation to be predicted (above cited papers and manuscript in preparation); (2) Transfer to/from a nascent protein is likely to follow associative ligand-exchange: This confers rapid on/off rates supporting the accuracy of such calculations; (3) Analogous calculations have been used to estimate the magnitude of ‘kinetic bias’ introduced by metallochaperone-mediated metalation (JACS Au 2023); (4) Ongoing research suggests that relative in vitro binding preferences of proteins that kinetically-trap metals at folding may be used in the calculator (manuscript in preparation). The calculator is a beta-version with testing ongoing (manuscript in preparation). We are keen to have feedback.
For more information and to learn about assumptions and constraints of the calculator contact [email protected]