An immune complex selective affinity matrix utilizing a synthetic peptide

A synthetic peptide possessing an amino acid sequence patterned on the globular head region of human complement component 1 subcomponent q (C1q) was tested for immunoglobulin binding. The peptide designated complementary binding peptide 2 (CBP2) was able to inhibit Staphylococcus aureus Protein A and human C1q from binding rabbit immunoglobulin at peptide concentrations for 50% inhibition of 1 and 10 microM, respectively. When attached to a solid-phase matrix in a column, CBP2 was able to bind immune complexes consisting of horseradish peroxidase plus rabbit antiperoxidase antibody or alkali-aggregated human immunoglobulins. A 1:4 mixture of immune complex to free immunoglobulin when passed over the CBP2 column demonstrated selective immune complex binding. Further controls established that CBP2 was in fact binding the immunoglobulin component of the immune complexes in a reversible fashion. The immune complex specificity of the column suggested a functional affinity was forming when CBP2 interacted with immune complexes. The possibility that the sequence of CBP2 is part of the immunoglobulin binding site of human C1q is discussed.     

Contribution of chondroitin sulfate A to the binding of complement proteins to activated platelets

Background

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.

Principal Findings

Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.

Conclusions

This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases.     

Mechanisms of stable serum resistance of Neisseria gonorrhoeae

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab)₂ isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab)₂ prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab)₂.Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab)₂ before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.     

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.     

The Mechanism of Inhibitory Effect of Polyanions and Polycations on the Activation of the Complement First Component

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 g/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 ± 6 and 8.1 ± 0.1 g/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 ± 3 g/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q–antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.     

A possible calcium binding site in D1 protein: A fluorescence and FTIR study of the interaction between lanthanides and a synthetic peptide

A peptide ranging from residue 229 to 240 of the D₁ protein of Photosystem (PS) II was synthesized and lanthanides were used as candidates of calcium. Fluorescence and FTIR spectroscopy were used to test the conformational adaptation after lanthanide additions. Fluorescence spectroscopy showed that the synthetic peptide provides lanthanide binding site, and that glutamic acids are involved in lanthanide binding. Resolution enhancement techniques were combined with band curve-fitting procedures to quantitate the FTIR spectral information from the amide 1 bands. The relative areas of these component bands indicate that lanthanide induced a substantial decrease in the amount of unordered structure and turns, while a corresponding increase in the amount of -helix and open loop was also observed. This indicates that a relatively compact structure of the synthetic peptide is formed if lanthanides are applied. The results may reflect on the physiological and biochemical function of calcium in PS II, including preventing D₁ from trypsin digestion.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - FTIR Fourier transform infrared - FSD Fourier self-deconvolution - PS Photosystem - Q_B Secondary plastoquinone electron acceptor of PS II     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

Background

Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated.

Methods and Findings

The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration.

Conclusion

Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9.     

Characterization of prostate-tissue-directed monoclonal antibody, α-Pro 13

Summary The -Pro 13-secreting hybridoma was produced by immunizing mice with an equal mixture of PC-3, DU145, and LNCaP established prostatic carcinoma cell lines. The specificity of -Pro 13 monoclonal antibody was evaluated by the criteria of differential binding to cultured cells; differential binding to extracts of malignant prostate, nonmalignant prostate, and malignant and nonmalignant tissues of various histiotypes in solid phase radioimmunoassay; and by immunoperoxidase staining of primary surgical tissues of varied histiotypes. The data generated by multiple assay investigation indicate that -Pro 13 exhibits preferential binding to the ductal epithelium of prostate tissue; immunoperoxidase evaluation indicates a considerable heterogeneity of staining of ductal epithelial cells. The most prevalent cross-reactivity of -Pro 13 monoclonal antibody with non-prostate tissue occurs with blood vessel endothelium of restricted tissues. Electrophoretic analysis of immunoprecipitates from radioiodinated prostatic tumor extracts indicates that the molecule recognized by -Pro 13 is of 120,000 dalton apparent nonreduced molecular weight. Under reducing conditions, the antigen (p40) consists of a major component of 40,000 dalton apparent MW and a minor component of 17,000 dalton MW. p40 has an isoelectric point of 3.5–4.5. The antigen is intrinsically stable on the PC-3 cell surface; its release into spent culture medium is negligible. p40 is also stable upon complexation with -Pro 13 antibody in that it is not shed from the cell surface as an immune complex nor is it endocytosed to any extent as an immune complex.Abbreviations MIg anti-mouse immunoglobulin - IEF isoelectric focusing - kD kilodalton - MW molecular weight - PA Staphylococcus protein A - RIA radioimmunoassay - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SCM spent culture medium     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

C1q and C4b bind simultaneously to CR1 and additively support erythrocyte adhesion.

Previously, we showed that soluble C1q bound specifically to CR1 on transfected cells. If the CR1-C1q interaction were to participate in immune complex clearance, then this interaction should support E adhesion. Using a tip plate adhesion assay, we found that immobilized C1q mediated adhesion of human E. E binding to C1q was specifically inhibited by polyclonal anti-CR1 Fab fragments. Intact C1 was not efficient as an adherence ligand until it was treated with EDTA or the C1 inhibitor to remove the C1r2C1s2 complex from C1, leaving C1q. Titration of C1q alone, C4b alone, and C1q + C4b indicated that the two complement ligands were additive in their ability to support CR1-mediated adhesion of E. Analysis of binding to immobilized CR1 using a BIAcore instrument documented that C1q, C4b, and C3b binding were independent events. Additionally, C1q-dependent binding of immune complexes and heat-aggregated IgG to E was documented. These experiments confirm that the immune adherence receptor in humans, CR1, is the single receptor for all of the opsonic ligands of complement, provide evidence for a single C1q binding site on LHR-D of CR1, and suggest that C1q may participate in immune clearance.     

Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca²⁺-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.     

Atrial natriuretic peptide binding sites in the mammalian heart: Localization to endomural vessels

Summary The distribution of binding sites for atrial natriuretic peptide in cardiac ventricles of several mammalian species, including rat and human, was determined by in vitro autoradiography. The results revealed a unique anatomic localization of atrial natriuretic peptide binding sites to endomural vessels (Thebesian vessels), which communicate directly with the ventricular chambers. Digital image analysis indicated that these vascular channels possessed binding site densities comparable to those of the renal glomeruli a major target site for circulating atrial natriuretic peptide. In contrast, no specific labeling of branches of the coronary arteries and veins was detected. The discrete localization of atrial natriuretic peptide binding sites to this primitive cardiac circulatory system allows speculation as to the role of this hormone in the regulation of endocardialcirculation during cardiac development, normal ventricular function, and in coronary insufficiency.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

A new antigenic epitope appears in the catalytic subunit of viscumin during intracellular transport

The plant toxin viscumin (60 kD) consists of B- (binding) and A- (active) subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody–toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody–toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of ¹H-NMR spectroscopy.     

The role of immune complexes in the activation of the first component of human complement

Immune complex-induced C1 activation and fluid phase C1 autoactivation have been compared in order to elucidate the immune complex role in the C1 activation process. Kinetic analyses revealed that immune complex-bound C1 activates seven times faster than fluid phase C1 spontaneously activates. The rate of spontaneous C1 activation increased after decreasing the solution ionic strength. In fact at one-half physiologic ionic strength (i.e., 0.08 M), the kinetics of spontaneous C1 activation were indistinguishable from the kinetics of activation of immune complex-bound C1 at physiologic ionic strength. The enhanced fluid phase C1 activation at low ionic strength resulted neither from C1 nor C1q aggregation, nor from selective effects on the C1r2S2 subunit; however, at the reduced ionic strength, the C1 association constant (defined for C1q + C1r2S2 in equilibrium C1qr2S2) did increase to 2.3 X 10(8) M-1, which is equal to that for C1 bound to an immune complex at physiologic ionic strength. Therefore, C1 can spontaneously activate in the fluid phase as rapidly as C1 on an immune complex when the strength of interaction between C1q and C1r2S2 is the same in both systems. In conclusion, under physiologic conditions, C1q and C1r2S2 are two weakly interacting proteins. Immune complexes provide a site for the assembly of a stable C1 complex, in which C1q and C1r2S2 remain associated long enough for C1q to activate C1r2S2. Thus, immune complexes enhance the intrinsic C1 autoactivation process by strengthening the association of C1q with C1r2S2.     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

Measurement of 2J(H,C)- and 3J(H,C)-coupling constants by α/β selective HC(C)H-TOCSY

A new heteronuclear NMR pulse sequence for the measurement of ⁿJ(C,H) coupling constants, the /selective HC(C)H-TOCSY, is described. It is shown that the S³E element (Meissner et al., 1997a,b) can be used to obtain spin state selective coherence transfer in molecules, in which adjacent CH moieties are labeled with ¹³C. Application of the / selective HC(C)H-TOCSY to a 10nt RNA tetraloop 5-CGCUUUUGCG-3, in which the four uridine residues are ¹³C labeled in the sugar moiety, allowed measurement of two bond and three bond J(C,H) coupling constants, which provide additional restraints to characterize the sugar ring conformation of RNA in cases of conformational averaging.     

DNA binds and activates complement via residues 14-26 of the human C1q A chain.

The mechanism by which DNA activates the classical complement pathway was investigated, with emphasis upon the C1q binding sites involved. DNA bound to both the collagen-like and globular regions of C1q. Binding reactivity with DNA was retained after reduction/alkylation and sodium dodecyl sulfate treatment of C1q. DNA bound preferentially to the A chain of C1q. Binding sites for DNA were localized by using synthetic C1q A chain peptides to two cationic regions within residues 14-26 and 76-92, respectively. Peptides 14-26 and 76-92 avidly bound DNA in enzyme-linked immunosorbent and gel shift assays. Peptide 14-26 also precipitated with DNA and blocked its ability to bind C1q and activate C. Replacement of the two prolines with alanines or scrambling the order of the amino acids resulted in loss of ability of peptide 14-26 to inhibit C1q binding and complement activation by DNA; similar investigations showed a sequence specificity for peptide 76-92 as well. These experiments identify C1q A chain residues 14-26 as the major site, and residues 76-92 as a secondary site, through which DNA binds C1q and activates the classical complement pathway, and demonstrate that a peptide identical to residues 14-26 can modulate C1q binding and complement activation by DNA.     

Synthesis of an iron(II)-braced proline-II tripod protein

This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed, chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (FeII(Mbc)3: Mbc is 4-methyl-2,2-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro5-Pra(Mbc)-Pro3-NH2: Pra is cis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling Mbc-OH to the 4-amino group of Boc-Pra-OCH3 and saponifying the methyl ester group.The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe2+ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and a negative –* band at 312 nm, so its FeII(Mbc)3 brace was predominantly in the configuration. In a mostly acetonitrile solution at 25 °C, the leg peptide and the unbraced tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained in the proline-II helical form.