1. Comparative Binding Energy (COMBINE) Analysis of Barnase-Barstar Interfacial Mutants barstar barnase High binding affinity (Kd=10 -14 M) Polar binding interface: positive(+2e) on BN, negative (-6e) on BS Hot-spots Residues at the interface Binding features:

2. 65 complexes with interfacial mutations Arg59 Glu73 Arg83 His102 Arg87 Lys27 Glu80 Glu76 Trp38 Thr42 Tyr29 Asp39 Asp35 Trp44 Barnase Barstar

3. 65 complexes with different interfacial mutations

5. Electrostatic Desolvation Free Energy Calculation UHBD6.1: Solving the Poisson-Boltzmann equation using a finite difference method

6. Electrostatic Eesolvation Energy of Barnase Electrostatic Eesolvation Energy of Barstar R 2 <0.1 R 2 =0.41

7. R 2 =0.63 R 2 =0.35 Electrostatic Interaction Energy between Barnase and Barstar Electrostatic Binding Free Energy

8. Energy Decomposition to each BN-BS residue pair Each complex was energy-minimized in AMBER7.0 with AMBER94 ff Lennard-Jones interactions were calculated for each BN-BS residue pair (110 x 89 = 9790) Coulomic interactions were calculated for each BN-BS residue pair (110 x 89 = 9790) Desolvation energies of BN and BS were calculated in UHBD6.1 Each complex: 19582 (=9790+9790+2) energy terms

9. Chemometric Analysis Principal Component Analysis (PCA) Partial Least Squares Analysis (PLS) Contribution of each energy term: Importance of each residue pair Contribution of translational and rotational entropy change

10. Data set #Complexes#LVR2R2 Q2Q2 SDEP (kcal/mol) Constant C (kcal/mol) GG 65 40.650.571.67-2.928 50.850.741.29+1.63 60.910.860.97+5.364 70.930.880.87+6.351 Table 1. Predictive Performance of the COMBINE Model

11. Normalized Coefficients at 6 LV BarstarBarnase