Table 3.
Summary of global thermodynamic stability, pKa values of internal Lys residues and effects of pH on the HSQC spectra of variants of SNase with internal Lys residues
| Variant | pKa of internal Lysa |
% peaks preserved from Δ+PHSb |
Exchange Typec |
# Exchanged Peaksd |
ΔG°H2Oe neutral (kcal/mol) |
ΔG°H2Oe ionized (kcal/mol) |
|---|---|---|---|---|---|---|
| G20K | 10.4 | 68% (57/84) | 7.5(10.0) | 9.2 (9.0) | ||
| V23K | 7.3 | 59% (70/119) | 6.2 (8.4) | 4.0 (6.0) | ||
| L25K | 6.3 | 78% (100/129) | Intermed. | ~50 | 4.6 (7.5) | 2.7 (5.5) |
| F34K | 7.1 | 59% (70/119) | Intermed. | ~24 | 5.2 (8.0) | 3.8 (6.0) |
| L36K | 7.2 | 70% (83/119) | 6.4 (8.4) | 3.8 (6.0) | ||
| L37K | 10.4 | 68% (54/79) | 7.9 (9.9) | 9.4 (8.5) | ||
| L38Kf | 10.4 | 75% (59/79) | 7.5 (9.9) | 8.9 (8.4) | ||
| V39K | 9.0 | 67% (68/101) | Intermed. | ~50 | 4.1(10.1) | 4.7 (7.5) |
| T41K | 9.3 | 55% (51/93) | 8.6(10.0) | 9.5 (8.0) | ||
| A58K | 10.4 | 66% (52/79) | Slowg | 6.6 (9.9) | 7.8 (8.5) | |
| T62K | 8.1 | 85% (86/101) | Slowh | 8.5 (8.9) | 7.7 (7.0) | |
| V66Kf | 5.6 | 71% (92/130) | Intermed. | ~20 | 4.2 (7.0) | 2.0 (4.4) |
| I72K | 8.6 | 87% (88/101) | 5.6 (9.8) | 5.5 (7.0) | ||
| V74K | 7.4 | 51% (57/112) | 5.6 (8.4) | 4.4 (6.5) | ||
| A90K | 8.6 | 46% (46/101) | 4.5 (9.9) | 4.5 (7.9) | ||
| Y91K | 9.0 | 42% (39/93) | 4.5 (9.9) | 5.1 (7.9) | ||
| I92Kf | 5.3 | 45% (59/130) | Slow | ~130 | 1.9 (6.5) | 0.2 (4.9) |
| V99K | 6.5 | 63% (75/119) | Slowh | 4.1 (7.9) | 2.0 (5.4) | |
| N100K | 8.6 | 27% (27/101) | Slow | ~101 | 1.5(10.0) | 1.3 (7.0) |
| L103K | 8.2 | 62% (69/112) | 6.7 (8.9) | 6.1 (7.0) | ||
| V104K | 7.7 | 63% (71/112) | 5.0 (8.9) | 3.6 (6.5) | ||
| A109K | 9.2 | 59% (55/93) | 7.1 (9.8) | 7.5 (9.5) | ||
| N118K | 10.4 | 81% (64/79) | 8.8 (9.8) | 9.4 (8.5) | ||
| L125K | 6.2 | 74% (96/130) | Intermed. | ~15 | 4.2 (7.0) | 2.5 (4.9) |
| A132K | 10.4 | 58% (49/84) | 4.6 (9.8) | 5.4 (8.4) |
pKa values are from (Isom et al., 2011)
Percentage of peaks assigned by visual inspection from the Δ+PHS background spectrum at a pH where internal Lys in the variant is > 90% neutral. Values in parentheses indicate the number of visually assigned peaks divided by the total number of assigned peaks in the background spectrum. Error is ± 5%.
Major form of chemical exchange observed in HSQC spectra collected as a function of pH
Owing to the inclusion of unassigned peaks in the count this number is just an approximation
ΔG°H2O (kcal/mol) refers to the Gibbs free energy of unfolding of the protein measured with GdnHCl denaturation and extrapolated to zero denaturant concentration as described in (Isom et al., 2008, 2011). ‘Neutral’ refers to the ΔG°H2O value at approximately 1 pH unit above the pKa of the internal Lys and ‘ionized’ refers to the value at approximately 1 pH unit below the pKa. The number in parentheses is the pH at which the measurement was made. Errors in ΔG°H2O are ± 0.1 kcal/mol.
Data for variants with Lys-66, Lys-38, Lys-92 were published previously (Harms et al., 2008; Nguyen et al., 2004; Stites et al., 1991).
Slow exchange between two closely related native forms. A small subset of resonances are exchanging between two folded conformers.
Slow exchange with a partially unfolded form that is only a minor component of the ensemble, as evidenced by the persistence of well-resolved folded peaks at the low pH endpoint of the titration.
See also Figures S1–S3 and Table S1.