Molecular interactions of the CTLA-4 cytoplasmic region with the phosphoinositide 3-kinase SH2 domains.

During T-cell regulation, T-cell receptors and CD28 lead to signaling activation, while T-lymphocyte antigen 4 (CTLA-4) is known to lead to downregulation, similar to programmed cell death-1 (PD-1). In the cytoplasmic tails of CD28 and CTLA-4, phosphoinositide 3-kinase (PI3K) binds to the consensus sequence including phosphotyrosine via SH2 domains, N- and C-terminal SH2 domains (nSH2 and cSH2), of its regulatory subunit, p85. In this study, we determined the crystal structure of a CTLA-4-derived phosphopeptide in complex with a Cys-substituted mutant of cSH2, C656S/C659V/C670L, at a 1.1 Å resolution. Phosphotyrosine of the bound peptide is tightly accommodated by the residues Arg631, Arg649, Ser651, and Ser652, similar to the cSH2 wild-type recognition mode of CD28, as reported previously. Upon the Cys mutation, the cSH2 thermal stability increased while the CTLA-4 binding affinity slightly changed. The binding experiments also showed that the binding affinity of CTLA-4 by cSH2 was approximately two orders of magnitude lower than that of CD28. Similar to CD28 binding, the CTLA-4 binding affinity of nSH2 was lower than that of cSH2. The complex structure of nSH2 and CTLA-4 was modeled, and compared with the crystal structure of cSH2 mutant and CTLA-4. The difference in the binding affinity between CD28 and CTLA-4, along with the difference between nSH2 and cSH2, could be explained by the 3D structures, which would be closely correlated with the respective T-cell signaling.

Structural and functional properties of Grb2 SH2 dimer in CD28 binding.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells. In this study, we purified the dimer and monomer forms of Grb2 SH2, respectively, and analyzed their structural and functional properties. Size exclusion chromatography analysis showed that both dimer and monomer exist as stable states. Thermal stability analysis using circular dichroism showed that the dimer mostly dissociates into the monomer around 50°C. CD28 binding experiments showed that the affinity of the dimer to the phosphopeptide was about three fold higher than that of the monomer, possibly due to the avidity effect. The present crystal structure analysis of Grb2 SH2 showed two forms; one is monomer at 1.15 Å resolution, which is currently the highest resolution analysis, and another is dimer at 2.00 Å resolution. In the dimer structure, the C-terminal region, comprising residues 123-152, was extended towards the adjacent molecule, in which Trp121 was the hinge residue. The stable dimer purified using size exclusion chromatography would be due to the C-terminal helix "swapping". In cases where a mutation caused Trp121 to be replaced by Ser in Grb2 SH2, this protein still formed dimers, but lost the ability to bind CD28.

Characterization of the Ca2+-coordination structures of L- and T-plastins in combination with their synthetic peptide analogs by FTIR spectroscopy.

FTIR spectroscopy was employed to characterize the coordination structures of divalent cations (M2+ = Ca2+ or Mg2+) bound by L- and T-plastins, which contain two EF-hand motifs. We focused on the N-terminal headpieces in the L- and T-plastins to analyze the regions of COO- stretching and amide-I in solution. The spectral profiles indicated that these headpieces have EF-hand calcium-binding sites because bands at 1551 cm-1 and 1555 cm-1 were observed for the bidentate coordination mode of Glu at the 12th position of the Ca2+-binding site of Ca2+-loaded L-plastin and T-plastin, respectively. The amide-I profile of the Mg2+-loaded L-plastin headpiece was identical with that of the apo L-plastin headpiece, meaning that L-plastin has a lower affinity for Mg2+. The amide-I profiles for apo, Mg2+-loaded and Ca2+-loaded T-plastin suggested that aggregation was generated in protein solution at a concentration of 1 mM. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of data obtained for synthetic peptide analogs corresponding to the Ca2+-binding site.

Coordination to Divalent Cations by Calcium-Binding Proteins.

Fourier-transform infrared spectroscopy (FTIR) is a powerful tool for examining the metal coordination of the side chain COO- groups of Glu and Asp on Ca2+-binding proteins in solution. The behavior of COO- symmetric stretch can be investigated by using protein samples in H2O solution. However, it is difficult to obtain information about the behavior of the COO- antisymmetric stretch in H2O solution, because the COO- antisymmetric stretching band overlaps with the amide II band. Therefore, to obtain reliable infrared spectra in the region of COO- antisymmetric stretch, exchangeable protons in the protein should be completely deuterated by incubating the apoprotein dissolved in D2O under mild heating conditions.

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

Quantitative analysis by surface plasmon resonance of CD28 interaction with cytoplasmic adaptor molecules Grb2, Gads and p85 PI3K.

CD28 surface receptors provide co-stimulatory signals that are required for full T cell activation. The CD28 cytoplasmic region has one YMNM and two PXXP motifs as a functional motif. Upon CD28 ligation, Grb2, Gads, and the p85 subunit of PI3 kinase are recruited to the CD28 cytoplasmic region. Here, the interactions between these adaptor proteins and CD28 cytoplasmic domains were analyzed using a Biacore surface plasmon resonance biosensor. For all three adaptor proteins, entire molecules bound more tightly to CD28 than did their isolated SH2 domains. For each adaptor, different outcomes of mutation of CD28's PXXP motifs on binding affinity indicated that only the SH3 domain of Grb2 bound directly. Regarding binding of SH2s to CD28, the SH2 domains of p85 bound more strongly than those of both Grb2 and Gads. Since intact p85 had a 50-fold higher binding affinity than its fragments, and yet the p85-CD28 interaction does not involve SH3-PXXP binding, binding of both N-terminal and C-terminal SH2s to YMNM may create an "avidity" effect. In contrast, when Grb2 and Gads interact with CD28, binding of their SH3 domains may be important. These results suggest that all these interactions are multivalent, through both SH2 and SH3 domains.

High resolution crystal structure of the Grb2 SH2 domain with a phosphopeptide derived from CD28.

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I ß-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I ß-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

Effects of the KIF2C neck peptide on microtubules: lateral disintegration of microtubules and ß-structure formation.

Members of the kinesin-13 sub-family, including KIF2C, depolymerize microtubules. The positive charge-rich 'neck' region extending from the N-terminus of the catalytic head is considered to be important in the depolymerization activity. Chemically synthesized peptides, covering the basic region (A182-E200), induced a sigmoidal increase in the turbidity of a microtubule suspension. The increase was suppressed by salt addition or by reduction of basicity by amino acid substitutions. Electron microscopic observations revealed ring structures surrounding the microtubules at high peptide concentrations. Using the peptide A182-D218, we also detected free thin straight filaments, probably protofilaments disintegrated from microtubules. Therefore, the neck region, even without the catalytic head domain, may induce lateral disintegration of microtubules. With microtubules lacking anion-rich C-termini as a result of subtilisin treatment, addition of the peptide induced only a moderate increase in turbidity, and rings and protofilaments were rarely detected, while aggregations, also thought to be caused by lateral disintegration, were often observed in electron micrographs. Thus, the C-termini are not crucial for the action of the peptides in lateral disintegration but contribute to structural stabilization of the protofilaments. Previous structural studies indicated that the neck region of KIF2C is flexible, but our IR analysis suggests that the cation-rich region (K190-A204) forms ß-structure in the presence of microtubules, which may be of significance with regard to the action of the neck region. Therefore, the neck region of KIF2C is sufficient to cause disintegration of microtubules into protofilaments, and this may contribute to the ability of KIF2C to cause depolymerization of microtubules.

Infrared study of synthetic peptide analogues of the calcium-binding site III of troponin C: The role of helix F of an EF-hand motif.

The EF-hand motif (helix-loop-helix) is a Ca(2+)-binding domain that is common among many intracellular Ca(2+)-binding proteins. We applied Fourier-transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E-loop-helix F). The 17-residue peptides corresponding to loop-helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D-type amino acid, were investigated to disturb the α-helical conformation of helix F systematically. These D-type-substituted peptides showed no band at about 1555 cm(-1) even in the Ca(2+)-loaded state although the native peptide (L-type only) showed a band at about 1555 cm(-1) in the Ca(2+)-loaded state, which is assigned to the side-chain COO(-) group of Glu at the 12th position, serving as the ligand for Ca(2+) in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca(2+) and the side-chain COO(-) group of Glu at the 12th position. Implications of the COO(-) antisymmetric stretch and the amide-I' of the synthetic peptide analogues of the Ca(2+)-binding sites are discussed.

Coordination to divalent cations by calcium-binding proteins studied by FTIR spectroscopy.

We review the Fourier-transform infrared (FTIR) spectroscopy of side-chain COO(-) groups of Ca(2+)-binding proteins: parvalbumins, bovine calmodulin, akazara scallop troponin C and related calcium binding proteins and peptide analogues. The COO(-) stretching vibration modes can be used to identify the coordination modes of COO(-) groups of Ca(2+)-binding proteins to metal ions: bidentate, unidentate, and pseudo-bridging. FTIR spectroscopy demonstrates that the coordination structure of Mg(2+) is distinctly different from that of Ca(2+) in the Ca(2+)-binding site in solution. The interpretation of COO(-) stretches is ensured on the basis of the spectra of calcium-binding peptide analogues. The implication of COO(-) stretches is discussed for Ca(2+)-binding proteins. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Extensively hydrated but folded: a novel state of globular proteins stabilized at high pressure and low temperature.

We studied conformational fluctuations of the transcription factor c-Myb R2 subdomain (52 residues with three Trp) at high pressure and low temperature (5°C) using two different spectroscopic methods, Trp fluorescence and (1)H NMR, on its chemically stable mutant C130I (pseudo-wild-type (WT(S))), which has a large internal cavity. As pressure was increased from 3 to 300 MPa, the Trp fluorescence λ(max) of WT(S) shifted from 342 to ∼355 nm, clearly showing that the three Trp rings become fully exposed to the polar environment, which usually is taken to indicate that the protein underwent unfolding. In contrast, as pressure was increased from 3 to 300 MPa, the high-field-shifted (1)H NMR signals characteristic of the folded state showed a still higher-field shift, but no significant changes in their intensity. The last result unequivocally shows that the protein remains largely folded at 300 MPa. The apparent discrepancy between the two predictions would only be solved if one were to postulate the existence of an extensively hydrated but folded state in WT(S). Intriguingly, such a state was not found in a cavity-filling mutant of WT(S), C130I/V103L, suggesting that this state is mediated by cavity hydration. The generality and significance of this state in proteins are discussed.

Stem-forming regions that are essential for the amyloidogenesis of prion proteins.

Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-ß-structure, the initial mechanism in the conversion to a ß-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-ß-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains ~200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular ß-sheet to a solvent through the partial transition of a native structure from an α-helix to a ß-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.

Effect of glycosylation on cis/trans isomerization of prolines in IgA1-hinge peptide.

The hinge region of human immunoglobulin A1 (IgA1), connecting the Fab and Fc regions, is mostly composed of Ser, Thr, and Pro (VPSTPPTPSPSTPPTPSPS); hinge peptide (HP). O-Glycans are naturally attached on only particular five Ser/Thr residues in this region. NMR was employed for analysis of the structural changes in HP upon the glycosylation, especially focusing on the cis/trans isomerization of Pro residues. A series of HP containing (13)C,(15)N-labeled Pro residues were chemically synthesized and enzymatically glycosylated. The signals from cis and trans forms of the labeled Pro were identified by two-dimensional NMR spectroscopy. Cis/trans ratios of the Pro residues at the C-terminal side of the glycosylated Ser/Thr were reduced from 9-10% to 2-3% by the glycosylation. Thermodynamic analyses indicated that the decrease in the cis/trans ratio was enthalpy-driven. Hydrogen-deuterium exchange experiments and NOE-based structure determination revealed that the intraresidue hydrogen bonds between the amide group of GalNAc and carbonyl oxygen of the peptide backbone of GalNAc-Thr are formed in the major trans conformers, which is consistent with the thermodynamic parameters. These hydrogen bonds largely restrict the psi angle of the peptide backbone and, thereby, should make the trans conformation of the C-terminal Pro residue more stable than the cis conformation. Namely, it is predicted that the restricted psi angle causes interresidue steric hindrance for the cis conformation. The appropriate glycosylation of HP probably contributes to the decrease in the unfavorable variety of relative orientations between Fab and Fc in IgA1, through stabilizing the conformation of HP.

Visible-light photocontrol of (E)/(Z) isomerization of the 4-(dimethylamino)azobenzene pseudo-nucleotide unit incorporated into an oligonucleotide and DNA hybridization in aqueous media.

We demonstrate significant photoresponsivity in aqueous media to visible light of pseudo-oligonucleotides possessing 4-(dimethylamino)azobenzene (4-DMAzo) side chains. The spectrum of the 4-DMAzo moiety during 436 nm light irradiation at pH >9 was clearly different from that of the all (E)-form, indicating the presence of the (Z)-form. Thermal (Z)-to-(E) recovery isomerization was faster at pH 9 (k(Z)(-E) = approximately 10(1) s(-1)) than at pH 11; however, addition of 50% ethanol significantly slowed the thermal recovery isomerization at pH 9 (k(Z)(-E) = approximately 2 s(-1)) and increased the magnitude of the spectral changes. Significant photoregulation of DNA hybridization by visible light was demonstrated under this condition.

The dynein stalk head, the microtubule binding-domain of dynein: NMR assignment and ligand binding.

Dynein is a motor ATPase, and the C-terminal two-thirds of its heavy chain form a ring structure. One of protrudings from this ring structure is a stalk whose tip, the dynein stalk head (DSH), is thought to be the microtubule-binding domain. As a first step toward elucidating the functional mechanisms of DSH, we aimed at the NMR structural analysis of an isolated DSH from mouse cytoplasmic dynein. The DSH expressed in bacteria and purified was coprecipitated with microtubules, suggesting its proper folding. Chemical shifts of the DSH were obtained from NMR measurements, and backbone assignment identified 94% of the main-chain N-H signals. Secondary structural prediction programs showed that about 60% of the residues formed alpha-helices. A region with cationic residues K58 and R61 (and possibly R66 as well), and another with R86, K88, K90, and K91, were found to form alpha-helices. Both of these regions may be important in the formation of the DSH-binding site to a microtubule that has a low pI with a number of acidic residues. Two synthetic peptides containing the sequence of the alpha-helix 12 of beta-tubulin, considered to be important in binding to DSH, were investigated. Of these two peptides, the one with higher helix-formation propensity appeared to bind to DSH, since it precipitated with DSH in a nearly stoichiometric manner. This suggested that the alpha-helicity of this region would be important in its binding to DSH.

Reversible and irreversible coiled coils in the stalk domain of ncd motor protein.

Ncd is a microtubule minus end-directed motor protein from Drosophila, a member of the kinesin-14 family, and an essential protein in mitosis and meiosis. Full-length ncd exists as a dimer via the formation of an alpha-helical coiled coil in its central stalk domain (P192-R346), which is thought to be one of the key regions for its motility. In our previous studies, however, none of the various synthetic polypeptide fragments (up to 46 residues) from the stalk domain formed a coiled coil. Herein, we have investigated the structural properties of the full-length ncd stalk domain using recombinant polypeptides together with shorter segments. These new fragments did form coiled coils as verified by far-UV circular dichroism (CD) spectroscopy and analytical ultracentrifugation, suggesting that a certain length of polypeptide would be required for dimer formation. Moreover, deletion mapping revealed that the cooperativity among the neighboring subdomains in the stalk domain is required for formation of the coiled coil. Interestingly, the intact stalk domain segments showed three-state transition in thermal unfolding measurements with CD, indicating the presence of two regions: (i) a coiled-coil region (P227-R306) that exhibits reversible denaturation at a lower temperature (20-30 degrees C) and (ii) a more rigid coiled-coil region (T307-E334) that exhibits irreversible denaturation at a high temperature (ca. 60 degrees C). These results imply that the N-terminal region of the stalk domain might be able to adopt both a coiled-coil conformation and a dissociated one, which might be relevant to the functions of ncd.

Interaction-based evaluation of the propensity for amyloid formation with cross-beta structure.

In order to reveal the requirements for amino acid sequences prone to form amyloid fibrils, a novel prediction method based on the original structural model of amyloids was developed. As a working hypothesis, two fundamental conditions were introduced into the design of the present system for the evaluation of the propensity for amyloidogenicity. The first of these two conditions was to ensure that the hydrophobic and hydrogen-bonding interactions between residues on neighboring antiparallel beta-strands were formed along a fibril axis. The other condition was that the hydrophobic interacting residues appeared on both faces of the protofibril, which gave line-matching interactions. Most peptides with sequences exhibiting high scores, as evaluated by this method, were found to easily form amyloids with the aid of a turn-inducing structure designed as a connection of two beta-strands. On the other hand, peptides with low-scoring native sequences and those modified by an internal residue-residue exchange (the latter yielding a null score) did not lead to amyloid formation. These data demonstrated the validity of this method for the prediction of amyloid structures. Moreover, the present study provided support for the proposed model of the essential structure associated with the above working hypothesis. The predicted high-scoring regions were in good agreement with the putative amyloid core regions reported thus far.

Coiled coil in the stalk region of ncd motor protein is nonlocally sustained.

The dimeric structure of kinesin superfamily proteins plays an important role in their motile functions and characteristics. In this study, the coiled-coil-forming property of the stalk region (192-346) of Drosophila ncd, a C-terminal kinesin motor protein, was investigated by synthesizing various peptide fragments. The alpha helicity of a set of 46-residue peptides spanning the stalk region appeared too low to form a coiled-coil dimer, probably because of insufficient continuity of the hydrophobic residues at (a and d) core positions in amphipathic heptad repeats. On the other hand, several peptides with leucine residues introduced at core positions or with extensional sequences with high alpha helicity had an advantage in coiled-coil formation. When we analyzed the thermal and urea-induced unfolding of these dimeric peptides, we identified four domains having a relatively high potential to form coiled coils. Among them, three domains on the C-terminal side of the stalk region, i.e., (252-272), (276-330), and (336-346), were in the same heptad frame, although these potential coiled-coil domains were not self-sustaining individually. This is in sharp contrast to the fragment of human kinesin, (332-369), which has an extremely high tendency toward coiled-coil formation. One of the possible triggers for coiled-coil formation of the ncd stalk region may be the interaction between the motor domain and the C-terminal part of the stalk as previously revealed by X-ray crystallography. The residues, S331 and R335, seem to act as a breaking point for alpha-helix continuity. This would make the region (336-346), as the head-stalk joint, more flexible such as seen with a plus-end-directed kinesin, if this region had no interaction with the motor domain. These characteristic differences between ncd and kinesin suggest that the nonlocally sustained coiled coil of ncd is one of the factors important for minus-end-directed motility.

Peripheral region for core cross-beta plays important role in amyloidogenicity.

The role of the peripheral sequence neighboring the core cross-beta region was investigated using a peptide library constructed with all possible combinations of Lys, Glu, Ser, and Leu at three residue positions (X1-X3) forming the N-terminal region linked to the amyloid core sequence of the barnase-derived segment (A4-K22). By means of CD spectra and thioflavin T binding assay for 64 peptides, not only the composition but also the sequence in the peripheral region were found to be responsible for amyloid formation. The preferences of amino acid residues in the peripheral region of the amyloid-forming peptides were in the order of Leu approximately SerGlu>>Lys. A balance of positive and negative charges was found to be essential for amyloid formation, suggesting that the electrostatic interaction at the surface of the amyloid fibrils is relevant to their stability. On the basis of the maximum fluorescence wavelength of fibril-bound thioflavin T, the highly amyloidogenic peptides were classified into two classes, which exhibited the sequence preferences of (Leu, Ser/Glu, and Leu) and (Glu, Leu, and Ser) for the peripheral sequence (X1, X2, and X3). The former class can be rationally assigned to the structural model with deep grooves along the fibril axis. Thus, the peripheral sequence regulates the manner of molecular packing in the fibrils as well as the amyloidogenicity. In addition, the chains of the peripheral sequence are most likely to form thioflavin T binding sites.

Fourier transform infrared spectroscopic study on the Ca2+ -bound coordination structures of synthetic peptide analogues of the calcium-binding site III of troponin C.

The coordination structures of Ca(2+) ion bound to synthetic peptide analogues of the calcium-binding site III of rabbit skeletal muscle troponin C (TnC) were investigated by Fourier transform infrared (FTIR) spectroscopy. The region of the COO(-) antisymmetric stretching vibration provides information about the coordination modes of a COO(-) group to a metal ion. The 34-residue peptide corresponding to the EF hand motif (helix-loop-helix) showed a band at 1552 cm(-1) in the Ca(2+)-loaded state, indicating that the side-chain COO(-) group of Glu at the 12th position serves as a ligand for Ca(2+) in the bidentate coordination mode. On the other hand, the 13-residue peptide (Ac-DRDADGYIDAEEL-NH(2)) containing the Ca(2+)-binding site III (DRDADGYIDAEE) did not show such spectral patterns in the Ca(2+)-loaded state, meaning that shorter synthetic peptide corresponding to the site III has less or no affinity for Ca(2+). It was found that the 17-residue peptide (Ac-DRDADGYIDAEELAEIF-NH(2)) is the minimum peptide necessary for the interaction of side-chain COO(-)of Glu at the 12th position with Ca(2+) in the bidentate coordination mode. We discuss the relationship between the amino acid length of synthetic peptide analogues and the formation of Ca(2+)-bound coordination structure.