Table 1. Summary of thermodynamic values determined for all barstar variants and srcSH3 in this study.
Equilibrium denaturant-induced unfolding transitions were performed in a single trial (n=1), and values reported represent fit parameters. Native-state proteolysis experiments were performed in triplicate (n=3; barstarK2, barstarK78, and barstarK60/E80A) or quadruplicate (n=4; barstarK60), and values represent the mean. All reported errors represent S.E.M derived from curve fitting and propagated through all calculations.
| BarstarK2 | BarstarK60 | BarstarK78 | BarstarK60/E80A | SH3 | |
|---|---|---|---|---|---|
| Cm unmodified (M urea) | 4.97 +/− 0.28 | 4.41 +/− 0.55 | 4.65 +/− 1.19 | 6.02 +/− 0.38 | n.d. |
| Cm monoUb (M urea) | 2.52 +/− 0.16 | 2.51 +/− 0.34 | 4.24 +/− 1.05 | 3.72 +/− 0.26 | n.d. |
| ΔCm (unmodified-monoUb) (M urea) | 2.45 +/− 0.32 | 1.90 +/− 0.65 | 0.42 +/− 1.59 | 2.30 +/− 0.46 | n.d. |
| m-valueglobal unmodified (kcal/mol M) | −1.06 +/− 0.04 | −0.96 +/− 0.08 | −0.89 +/− 0.17 | −1.16 +/− 0.05 | n.d. |
| m-valueglobal monoUb (kcal/mol M) | −0.59+/− 0.02 | −0.68 +/− 0.07 | −0.48 +/− 0.09 | −0.70 +/− 0.04 | n.d. |
| Δm-valueglobal unmodified-monoUb (kcal/mol M) | −0.47 +/− 0.05 | −0.28 +/− 0.11 | −0.41 +/− 0.19 | −0.47 +/− 0.06 | n.d. |
| ΔGunfolding Unmodified# (kcal/mol) | −5.27 +/− 0.20 | −4.25 +/− 0.38 | −4.16 +/− 0.74 | −6.99 +/− 0.31 | n.d. |
| ΔΔGunfolding unmodified-monoUb* (kcal/mol) | −2.02 +/− 0.10 | −1.56 +/− 0.22 | −0.28 +/− 0.45 | −2.14 +/− 0.11 | n.d. |
| ΔGproteolysis unmodified (kcal/mol) | −2.72 +/− 0.02 | −3.24 +/− 0.11 | −2.72 +/− 0.06 | −3.52 +/− 0.01 | −2.70 +/− 0.12 |
| ΔGproteolysis monoUb (kcal/mol) | −1.66 +/− 0.10 | −2.12 +/− 0.06 | −2.66 +/− 0.09 | −2.56 +/− 0.08 | −2.38 +/− 0.23 |
| ΔΔGproteolysis unmodified-monoUb (kcal/mol) | −1.07 +/− 0.11 | −1.12 +/− 0.13 | −0.06 +/− 0.15 | −0.96 +/− 0.12 | −0.32 +/− 0.10 |
#
indicates that ΔGunfolding values were calculated using a two-state model with linear extrapolation.
*
indicates that ΔΔGunfolding values were calculated by multiplying the Cm from the denaturation curves by the average m-value for the unmodified and mono-ubiquitinated proteins.