Distances between pairs of introduced residues and electrostatic ΔG values based on the K2PØ model
| Mutation | Distance to S104 or T216 Cβ–Cβ (ɛN-C) | ΔΔG versus WT |
| Å | kcal/mol | |
| Second site mutations that complement | ||
| Monomer subunits | ||
| S104K alone | 7.9 | |
| +N133D | 7.6 (3) | 0.3 |
| +G134D | 11.1 (10) | 5.2 |
| +F137D | 12.0 (8) | 1.3 |
| +A138D | 16.2 (15) | 5.9 |
| +V258D | 12.9 (7) | 0.8 |
| +T262D | 18.7 (16) | 6.5 |
| T216K alone | 8.7 | |
| +L257D | 8.8 (4) | 0.5 |
| +V258D | 13.1 (10) | 3.9 |
| +M261D | 12.4 (8) | 1.2 |
| +T262D | 17.5 (15) | 6.7 |
| +G134D | 14.6 (11) | 4.4 |
| +A138D | 19.0 (15) | 4.8 |
| +G139D | 22.8 (21) | 7.4 |
| S104E alone | 6.9 | |
| +F137K | 12.0 (4) | 1.1 |
| +V258K | 12.9 (7) | 1.6 |
| +V258R | 12.9 (4) | 1.8 |
| Tandem subunits | ||
| S104K (I) alone | 4.1 | |
| +N133D (I) | 7.6 (3) | 0.5 |
| +V258D (II) | 12.9 (10) | 2.3 |
| +V258D (IV) | 20.0 (15) | 3.5 |
| T216K (II) alone | 5.1 | |
| +G134D (I) | 15.8 (13) | 3.1 |
| +L257D (II) | 8.8 (7) | 0.5 |
| +G134D (III) | 14.6 (12) | 3.1 |
| Second site mutations that do not complement | ||
| Monomer subunits | ||
| S104K alone | 7.9 | |
| +P131D | 13.3 (13) | 8.2 |
| +V132D | 11.6 (13) | 8.4 |
| +I135Da | 15.2 (16) | 7.1 |
| +L136D | 13.4 (15) | 7.9 |
| +G139Da | 18.2 (19) | 7.2 |
| +G255D | 13.1 (15) | 7.6 |
| +Y256D | 16.2 (17) | 8.3 |
| +L257D | 12.8 (12) | 7.9 |
| +M259D | 17.8 (17) | 7.8 |
| +I260D | 19.2 (18) | 7.8 |
| +M261Da | 15.9 (12) | 3.9 |
| +F263Da | 22.4 (21) | 7.3 |
| T216K alone | 8.7 | |
| +G255D | 14.2 (17) | 8.4 |
| +Y256D | 13.1 (15) | 8.1 |
| +M259D | 16.0 (17) | 7.7 |
| +I260D | 13.9 (14) | 7.6 |
| +F263Da | 18.3 (19) | 6.9 |
| +P131D | 12.8 (12) | 8.3 |
| +V132D | 15.5 (14) | 7.2 |
| +N133D | 14.8 (11) | 8.5 |
| +I135Da | 18.2 (16) | 7.0 |
| +L136D | 20.3 (18) | 8.3 |
| +F137D | 15.8 (13) | 7.2 |
| S104E alone | 6.9 | |
| +F137Ra | 12.0 (4) | 1.2 |
| Tandem subunits | ||
| S104K (I) alone | 4.1 | |
| +L257D (II) | 12.8 (13) | 3.6 |
| +N133D (III) | 20.1 (15) | 5.0 |
| +L257D (IV) | 18.4 (19) | 4.2 |
| T216K (II) alone | 5.1 | |
| +N133D (I) | 15.8 (13) | 4.1 |
| +N133D (III) | 14.8 (11) | 4.7 |
| +L257D (IV) | 20.8 (16) | 4.0 |
| Mutation | Distance to S104 or T216 Cβ–Cβ (ɛN-C) | ΔΔG versus WT |
| Å | kcal/mol | |
| Second site mutations that complement | ||
| Monomer subunits | ||
| S104K alone | 7.9 | |
| +N133D | 7.6 (3) | 0.3 |
| +G134D | 11.1 (10) | 5.2 |
| +F137D | 12.0 (8) | 1.3 |
| +A138D | 16.2 (15) | 5.9 |
| +V258D | 12.9 (7) | 0.8 |
| +T262D | 18.7 (16) | 6.5 |
| T216K alone | 8.7 | |
| +L257D | 8.8 (4) | 0.5 |
| +V258D | 13.1 (10) | 3.9 |
| +M261D | 12.4 (8) | 1.2 |
| +T262D | 17.5 (15) | 6.7 |
| +G134D | 14.6 (11) | 4.4 |
| +A138D | 19.0 (15) | 4.8 |
| +G139D | 22.8 (21) | 7.4 |
| S104E alone | 6.9 | |
| +F137K | 12.0 (4) | 1.1 |
| +V258K | 12.9 (7) | 1.6 |
| +V258R | 12.9 (4) | 1.8 |
| Tandem subunits | ||
| S104K (I) alone | 4.1 | |
| +N133D (I) | 7.6 (3) | 0.5 |
| +V258D (II) | 12.9 (10) | 2.3 |
| +V258D (IV) | 20.0 (15) | 3.5 |
| T216K (II) alone | 5.1 | |
| +G134D (I) | 15.8 (13) | 3.1 |
| +L257D (II) | 8.8 (7) | 0.5 |
| +G134D (III) | 14.6 (12) | 3.1 |
| Second site mutations that do not complement | ||
| Monomer subunits | ||
| S104K alone | 7.9 | |
| +P131D | 13.3 (13) | 8.2 |
| +V132D | 11.6 (13) | 8.4 |
| +I135Da | 15.2 (16) | 7.1 |
| +L136D | 13.4 (15) | 7.9 |
| +G139Da | 18.2 (19) | 7.2 |
| +G255D | 13.1 (15) | 7.6 |
| +Y256D | 16.2 (17) | 8.3 |
| +L257D | 12.8 (12) | 7.9 |
| +M259D | 17.8 (17) | 7.8 |
| +I260D | 19.2 (18) | 7.8 |
| +M261Da | 15.9 (12) | 3.9 |
| +F263Da | 22.4 (21) | 7.3 |
| T216K alone | 8.7 | |
| +G255D | 14.2 (17) | 8.4 |
| +Y256D | 13.1 (15) | 8.1 |
| +M259D | 16.0 (17) | 7.7 |
| +I260D | 13.9 (14) | 7.6 |
| +F263Da | 18.3 (19) | 6.9 |
| +P131D | 12.8 (12) | 8.3 |
| +V132D | 15.5 (14) | 7.2 |
| +N133D | 14.8 (11) | 8.5 |
| +I135Da | 18.2 (16) | 7.0 |
| +L136D | 20.3 (18) | 8.3 |
| +F137D | 15.8 (13) | 7.2 |
| S104E alone | 6.9 | |
| +F137Ra | 12.0 (4) | 1.2 |
| Tandem subunits | ||
| S104K (I) alone | 4.1 | |
| +L257D (II) | 12.8 (13) | 3.6 |
| +N133D (III) | 20.1 (15) | 5.0 |
| +L257D (IV) | 18.4 (19) | 4.2 |
| T216K (II) alone | 5.1 | |
| +N133D (I) | 15.8 (13) | 4.1 |
| +N133D (III) | 14.8 (11) | 4.7 |
| +L257D (IV) | 20.8 (16) | 4.0 |
Values for monomer subunits and those linked in tandem were determined (see Materials and methods). The presented distances are between Cβ–Cβ in the WT model and the closest approach of the ɛ-amino of suppressive lysine and carboxylate carbon of restoring aspartate in mutant models (ɛN-C). ΔΔG values are the difference between WT and mutant for the electrostatic free energy barrier that a potassium ion has to overcome traveling from the intracellular solution to the selectivity filter. pKa calculations predict that all second site changes that complement are charged.
Seven second site mutants are predicted to be charged but do not complement; five of these do not appreciably lower ΔΔG: F137R is predicted to rescue, but might be too close to S104E, resulting in steric hindrance, because two large residues cannot be accommodated in the available space. Discrepancy between experimental and computational results on rescue of S104K by M261D might indicate inaccuracy of the model.