Structure‐kinetic relationship analysis of the therapeutic complement inhibitor compstatin

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 × 10⁵ M^?1 s^?1) and a k_off variation of >35‐fold (1–37 × 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 β‐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. Copyright © 2009 John Wiley & Sons, Ltd.     

Structure of compstatin in complex with complement component C3c reveals a new mechanism of complement inhibition

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.     

The structural basis of compstatin activity examined by structure-function-based design of peptide analogs and NMR

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.     

A simple, yet highly accurate, QSAR model captures the complement inhibitory activity of compstatin

Compstatin is a 13-residue cyclic peptide inhibitor of complement activation that was originally identified through phage-mediated presentation of a peptide library to C3b. Recent efforts to improve its activity have led to a rich dataset of complement analogs, with the most active analog being approximately 260 times more active than the parent compstatin. In the present work, a highly transparent quantitative structure-activity relationship model (Radj2=0.89) with four parameters is presented that captures important physico-chemical and geometrical properties of the analog molecules with regard to activity. The number of aromatic bonds and hydrophobicity of the fourth residue of compstatin correlated strongly with activity. Also important were the hydrophobic patch size near the disulfide bond and the solvent-accessible surface area occupied by nitrogen atoms of basic amino acid residues.     

Species specificity of the complement inhibitor compstatin investigated by all‐atom molecular dynamics simulations

The development of compounds to regulate the activation of the complement system in non‐primate species is of profound interest because it can provide models for human diseases. The peptide compstatin inhibits protein C3 in primate mammals and is a potential therapeutic agent against unregulated activation of complement in humans but is inactive against nonprimate species. Here, we elucidate this species specificity of compstatin by molecular dynamics simulations of complexes between the most potent natural compstatin analog and human or rat C3. The results are compared against an experimental conformation of the human complex, determined recently by X‐ray diffraction at 2.4‐Å resolution. The human complex simulations provide information on the relative contributions to stability of specific C3 and compstatin residues. In the rat simulations, the protein undergoes reproducible conformational changes, which eliminate or weaken specific interactions and reduce the complex stability. The simulation insights can be used to design improved compstatin‐based inhibitors for human C3 and active inhibitors against lower mammals. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.

To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

A comprehensive calorimetric investigation of an entropically driven T cell receptor-peptide/major histocompatibility complex interaction

The alphabeta T cell receptor (TCR) is responsible for recognizing peptides bound and "presented" by major histocompatibility complex (MHC) molecules. We recently reported that at 25 degrees C the A6 TCR, which recognizes the Tax peptide presented by the class I MHC human leukocyte antigen-A*0201 (HLA-A2), binds with a weak DeltaH degrees , a favorable DeltaS degrees , and a moderately negative DeltaC(p). These observations were of interest given the unfavorable binding entropies and large heat capacity changes measured for many other TCR-ligand interactions, suggested to result from TCR conformational changes occurring upon binding. Here, we further investigated the A6-Tax/HLA-A2 interaction using titration calorimetry. We found that binding results in a pK(a) shift, complicating interpretation of measured binding thermodynamics. To better characterize the interaction, we measured binding as a function of pH, temperature, and buffer ionization enthalpy. A global analysis of the resulting data allowed determination of both the intrinsic binding thermodynamics separated from the influence of protonation as well as the thermodynamics associated with the pK(a) shift. Our results indicate that intrinsically, A6 binds Tax/HLA-A2 with a very weak DeltaH degrees , an even more favorable DeltaS degrees than previously thought, and a relatively large negative DeltaC(p). Comparison of these energetics with the makeup of the protein-protein interface suggests that conformational adjustments are required for binding, but these are more likely to be structural shifts, rather than disorder-to-order transitions. The thermodynamics of the pK(a) shift suggest protonation may be linked to an additional process such as ion binding.     

Heat capacity effects of water molecules and ions at a protein-DNA interface

The interaction of the TATA-box binding protein from the thermophilic and halophilic archaea Pyrococcus woesei (PwTBP) with an oligonucleotide containing a specific binding site is stable over a very broad range of temperatures and ionic strengths, and is consequently an outstanding system for characterising general features of protein-DNA thermodynamics. In common with other specific protein-DNA recognition events, the PwTBP-TATA box interaction is accompanied by a large negative change in heat capacity (ΔC_p) arising from the total change in solvation that occurs upon binding, which in this case involves a net uptake of cations. Contrary to previous hypotheses, we find no overall effect of ionic strength on this heat capacity change. We investigate the local contributions of site-specific ion and water binding to the overall change in heat capacity by means of a series of site-directed mutations of PwTBP. We find that although changes in the local ion binding capacity affect the enthalpic and entropic contributions to the free energy of the interaction, they do not affect the change in heat capacity. In contrast, we find remarkably large heat capacity effects arising from two particular symmetry-related mutations. The great magnitude of these effects is not explicable in terms of current semi-empirical models of heat capacity change. Previously reported X-ray crystal structures show that these mutated residues are at the centre of an evolutionarily conserved network of water-mediated hydrogen bonds between the protein and the DNA backbone. Consequently, we conclude that, in addition to water molecules buried in the protein-DNA interface that have been previously shown to influence heat capacity, bridging water molecules in a highly polar surface environment can also contribute substantially to negative heat capacity change on formation of a protein-DNA complex.     

New Compstatin Variants through Two De Novo Protein Design Frameworks

Two de novo protein design frameworks are applied to the discovery of new compstatin variants. One is based on sequence selection and fold specificity, whereas the other approach is based on sequence selection and approximate binding affinity calculations. The proposed frameworks were applied to a complex of C3c with compstatin variant E1 and new variants with improved binding affinities are predicted and experimentally validated. The computational studies elucidated key positions in the sequence of compstatin that greatly affect the binding affinity. Positions 4 and 13 were found to favor Trp, whereas positions 1, 9, and 10 are dominated by Asn, and position 11 consists mainly of Gln. A structural analysis of the C3c-bound peptide analogs is presented.     

Design of a modified mouse protein with ligand binding properties of its human analog by molecular dynamics simulations: the case of C3 inhibition by compstatin

The peptide compstatin and its derivatives inhibit the complement-component protein C3 in primate mammals and are potential therapeutic agents against the unregulated activation of complement in humans, but are inactive against C3 from lower mammals. Recent molecular dynamics (MD) simulations showed that the most potent compstatin analog comprised entirely of natural amino acids (W4A9) had a smaller affinity for rat C3, due to reproducible changes in the rat protein structure with respect to the human protein, which eliminated or weakened specific protein-ligand interactions seen in the human C3:W4A9 complex. Here, we study by MD simulations three W4A9 complexes with the mouse C3 protein, and two "transgenic" mouse derivatives, containing a small number (6-9) of human C3 substitutions. The mouse complex experiences the conformational changes and affinity reduction of the rat complex. In the "transgenic" complexes, the conformation remains closer to that of the human complex, the protein-ligand interactions are improved, and the affinity for compstatin becomes "human-like." The present work creates new avenues for a compstatin-sensitive animal model. A similar strategy, involving the comparison of a series of complexes by MD simulations, could be used to design "transgenic" sequences in other systems.     

Immobilization of arginase and its application in an enzymatic chromatographic column: thermodynamic studies of nor-NOHA/arginase binding and role of the reactive histidine residue

A biochromatographic approach is developed to measure for the first time changes in enthalpy, heat capacity change and protonation for the binding of nor-NOHA to arginase in a wide temperature range. For this, the arginase enzyme was immobilized on a chromatographic support. It was established that this novel arginase column was stable during an extended period of time. The affinity of nor-NOHA to arginase is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggested that the protonated group in the nor-NOHA-arginase complex exhibits a heat protonation of approximately -33 kJ/mol. This value agrees with the protonation of an imidazole group. Our result confirmed that active-site residue Hist 141 is protonated as imidazolium cation. Hist 141 can function as a general acid to protonate the leaving amino group of L-ornithine during catalysis. The thermodynamic data showed that nor-NOHA-arginase binding, for low temperature (<15 degrees C), is enthalpically unfavourable and being dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge-charge interactions contribute to the nor-NOHA-arginase complex formation. The temperature dependence of the free energy of binding is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, DeltaC(p)=-2.43 kJ/mol/K, of arginase. Above 15 degrees C, the thermodynamic data DeltaH and DeltaS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong enzyme-inhibitor hydrogen bond networks. As well, by the use of these thermodynamic data and known correlations it was clearly demonstrated that the binding of nor-NOHA to arginase produces slight conformational changes in the vicinity of the active site. Our work indicated that our biochromatographic approach could soon become very attractive for studying other enzyme-ligand binding.     

RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi

The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events.     

Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition.

The binding of a series of low molecular weight ligands towards trypsin and thrombin has been studied by isothermal titration calorimetry and protein crystallography. In a series of congeneric ligands, surprising changes of protonation states occur and are overlaid on the binding process. They result from induced pK(a) shifts depending on the local environment experienced by the ligand and protein functional groups in the complex (induced dielectric fit). They involve additional heat effects that must be corrected before any conclusion on the binding enthalpy (DeltaH) and entropy (DeltaS) can be drawn. After correction, trends in both contributions can be interpreted in structural terms with respect to the hydrogen bond inventory or residual ligand motions. For all inhibitors studied, a strong negative heat capacity change (DeltaC(p)) is detected, thus binding becomes more exothermic and entropically less favourable with increasing temperature. Due to a mutual compensation, Gibbs free energy remains virtually unchanged. The strong negative DeltaC(p) value cannot solely be explained by the removal of hydrophobic surface portions of the protein or ligand from water exposure. Additional contributions must be considered, presumably arising from modulations of the local water structure, changes in vibrational modes or other ordering parameters. For thrombin, smaller negative DeltaC(p) values are observed for ligand binding in the presence of sodium ions compared to the other alkali ions, probably due to stabilising effects on the protein or changes in the bound water structure.     

Binding of berberine to human telomeric quadruplex - spectroscopic, calorimetric and molecular modeling studies

This study examines the characteristics of binding of berberine to the human telomeric d[AG(3)(T(2)AG(3))(3)] quadruplex. By employing UV-visible spectroscopy, fluorescence spectroscopy and isothermal titration calorimetry, we found that the binding affinity of berberine to the human telomeric quadruplex is 10(6). The complete thermodynamic profile for berberine binding to the quadruplex, at 25 degrees C, shows a small negative enthalpy (DeltaH) of -1.7 kcal.mol(-1), an entropy change with TDeltaS of +6.5 kcal.mol(-1), and an overall favorable free energy (DeltaG) of -8.2 kcal.mol(-1) .Through the temperature dependence of DeltaH, we obtained a heat capacity (DeltaC(p)) of -94 (+/- 5) cal.mol(-1).K(-1). The osmotic stress method revealed that there is an uptake of 13 water molecules in the complex relative to the free reactants. Furthermore, the molecular modeling studies on different quadruplex-berberine complexes show that berberine stacking at the external G-quartet is mainly aided by the pi-pi interaction and the stabilization of the high negative charge density of O6 of guanines by the positively charged N7 of berberine. The theoretical heat capacity (DeltaC(p)) values for quadruplex-berberine models are -89 and -156 cal.mol(-1).K(-1).     

Studies of structure-activity relations of complement inhibitor compstatin

Compstatin, a 13-mer cyclic peptide, is a novel and promising inhibitor of the activation of the complement system. In our search for a more active analog and better understanding of structure-functions relations, we designed a phage-displayed random peptide library based on previous knowledge of structure activity relations, in which seven amino acids deemed necessary for structure and activity were kept fixed while the remaining six were optimized. Screening of this library against C3 identified four binding clones. Synthetic peptides corresponding to these clones revealed one analog, called acetylated Ile(1)Leu/His(9)Trp/Thr(13)Gly triple replacement analog of compstatin corresponding to clone 640 (Ac-I1L/H9W/T13G), which was more active than compstatin. This newly identified peptide had 4-fold higher activity when compared with the originally isolated form of compstatin and 1.6-fold higher activity when compared with acetylated compstatin (Ac-compstatin). The structures of Ac-I1L/H9W/T13G and Ac-compstatin were studied by nuclear magnetic resonance, compared with the structure of compstatin, and found to be very similar. The binding of Ac-I1L/H9W/T13G and the equally active acetylated analog with His(9)Ala replacement (Ac-H9A) to C3 was evaluated by surface plasmon resonance, which suggested similarity in their binding mechanism but difference when compared with Ac-compstatin. Compensatory effects of flexibility outside the beta-turn and tryptophan ring stacking may be responsible for the measured activity increase in Ac-I1L/H9W/T13G and acetylated analog with His(9)Ala replacement and the variability in binding mechanism compared with Ac-compstatin. These data demonstrate that tryptophan is a key amino acid for activity. Finally, the significance of the N-terminal acetylation was examined and it was found that the hydrophobic cluster at the linked termini of compstatin is essential for binding to C3 and for activity.     

Binding of aromatic donor molecules to lactoperoxidase: proton NMR and optical difference spectroscopic studies

The interaction of aromatic donor molecules with lactoperoxidase (LPO) was studied using 1H-NMR and optical difference spectroscopy techniques. pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with pKa of 6.1) which is presumably a distal histidine. Dissociation constants evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements were found to be an order of magnitude larger than those for binding to horse radish peroxidase (HRP), indicating relatively weak binding of the donors to LPO. The dissociation constants evaluated in presence of excess of I- and SCN- showed a considerable increase in their values, indicating that the iodide and thiocyanate ions compete for binding at the same site. The dissociation constant of the substrate binding was, however, not affected by cyanide binding to the ferric centre of LPO. All these results indicate that the organic substrates bind to LPO away from the ferric center. Comparison of the dissociation constants between the different substrates suggested that hydrogen bonding of the donors with the distal histidine amino acid, and hydrophobic interaction between the donors and the active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the LPO-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative and relatively low compared to those for binding to HRP. The distances of the substrate protons from the ferric center were found to be in the range 9.4-11.1 A which are 2-3 A larger than those reported for the HRP-substrate complexes. These structural informations suggest that the heme in LPO may be more deeply buried in the heme crevice than that in the HRP.     

Expression of compstatin in Escherichia coli: incorporation of unnatural amino acids enhances its activity

Compstatin, a 13-residue cyclic peptide, is a complement inhibitor that shows therapeutic potential. Several previous approaches have improved the activity of this peptide several-fold. In the present study, we have expressed and purified compstatin from Escherichia coli in an effort to increase its potency and to generate it in high yield in a more economical fashion. An intein-based expression system was used to express compstatin in fusion with chitin-binding domain and Ssp DnaB intein, which were later cleaved from the expressed molecule at room temperature and pH 7.0 to yield pure compstatin in one step. The expressed compstatin showed activity similar to the synthetic compstatin in an ELISA-based assay. The same expression system and purification strategy were used to incorporate three tryptophan analogs, 6-fluoro-tryptophan, 5-hydroxy-tryptophan, and 7-aza-tryptophan, into compstatin. Interestingly, incorporation of 6-fluoro-tryptophan increased the activity three-fold relative to wild-type compstatin; in contrast, incorporation of 5-hydroxy- or 7-aza-tryptophan rendered compstatin less active than the wild-type form.