Compstatin is a 13-residue peptide that inhibits activation of the complement system by binding to the central component C3 and its fragments C3b and C3c. A combination of theoretical and experimental approaches has previously allowed us to develop analogs of the original compstatin peptide with up to 264-fold higher activity; one of these analogs is now in clinical trials for the treatment of age-related macular degeneration (AMD). Here we used functional assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to assess the effect of modifications at three key residues (Trp-4, Asp-6, Ala-9) on the affinity and activity of compstatin and its analogs, and we correlated our findings to the recently reported co-crystal structure of compstatin and C3c. The K(D) values for the panel of tested analogs ranged from 10(-6) to 10(-8) M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4-fold range in k(on) values (2-10 x 10(5) M(-1) s(-1)) and a k(off) variation of >35-fold (1-37 x 10(-2) s(-1)) being observed. The stability of the C3b-compstatin complex seemed to be highly dependent on hydrophobic effects at position 4, and even small changes at position 6 resulted in a loss of complex formation. Induction of a beta-turn shift by an A9P modification resulted in a more favorable entropy but a loss of binding specificity and stability. The results obtained by the three methods utilized here were highly correlated with regard to the activity/affinity of the analogs. Thus, our analyses have identified essential structural features of compstatin and provided important information to support the development of analogs with improved efficacy.