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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.

that L-plastin is suitable for dynamic rearrangement of cytoskeletons, while T-plastin is suitable for maintaining static cytoskeletons 9 .
In the present study, Fourier transform infrared (FTIR) spectroscopy was employed to study the coordination structures of the divalent cation (M 2+ = Mg 2+ or Ca 2+ ) bound in mice L-and T-plastin headpieces, each of which contains two EF-hand Ca 2+ -binding sites (Fig. 1b) 9 . The regions of COO − antisymmetric and symmetric stretches provide information regarding the modes of coordination of a COO − group to a metal ion [10][11][12][13][14][15][16] . The results showed that these headpieces have Ca 2+ affinity in common with EF-hand calcium binding sites and a lower affinity for Mg 2+ . The amide-I profiles suggested that the T-plastin headpiece was more aggregable than the L-plastin headpiece. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of the data obtained for the synthetic peptide analogs corresponding to a Ca 2+ -binding site.

Results and Discussion
FtIR spectra of L-plastin headpiece. Figure 2a shows the FTIR spectra for an L-plastin headpiece in the apo (M 2+ -free) and Mg 2+ and Ca 2+ -loaded states in D 2 O solution in the wavenumber range of 1800-1300 cm −1 . We observed the bands due to amide-I' , COO − antisymmetric stretch, amide-II' , and COO − symmetric stretch from the higher wavenumber side in Fig. 2a(A-C). A slight difference was detected in the region  www.nature.com/scientificreports www.nature.com/scientificreports/ of COO − antisymmetric stretching: a shoulder at approximately 1551 cm −1 appeared in the Ca 2+ -loaded state, although such a shoulder was not observed in the apo and Mg 2+ -loaded states. Figure 2b also shows the FTIR spectra for the samples in H 2 O solution, where the contribution of the buffer has already been eliminated by subtracting the spectrum of the buffer, as mentioned in the Experimental section. The spectra indicated bands due to amide-I, amide-II, CH 2 bending, and COO − symmetric stretching from the higher wavenumber side. For the spectra obtained in H 2 O solution, the COO − antisymmetric stretching mode is overlapped with the amide II mode, and therefore, we cannot extract the information about the COO − antisymmetric stretch. However, the band at 1423 cm −1 was clearly observed only in the Ca 2+ -loaded state, which was thought to reflect the interaction of Ca 2+ with the side-chain COO − groups. The second-derivative spectra provide information in more detail regarding the spectral differences.
In Fig. 3, the second-derivative spectra corresponding to the data shown in Fig. 2 are shown. From the region of the COO − antisymmetric stretch, information regarding the coordination modes of the COO − groups to the metal ions such as bidentate or pseudo-bridging modes is obtained [10][11][12][13][14][15][16][17] . The bands at 1583 cm −1 and 1564 cm −1 in the apo as well as Mg 2+ -loaded states ( Fig. 3a(A,B)) were very close to the band at 1585 cm −1 due to β-COO − of Asp and the band at 1565 cm −1 due to γ-COO − of Glu, respectively [14][15][16] . The band at 1612 cm −1 was slightly stronger in the Mg 2+ -loaded state in comparison with that in the apo state. We observed the bands at 1604, 1580, and 1551 cm −1 in the Ca 2+ -loaded state where the band at 1551 cm −1 was undoubtedly due to the sidechain COO − group binding to Ca 2+ in the bidentate coordination mode 15,16 . In the COO − symmetric stretch region, two bands at 1423 and 1403 cm −1 were detected, although one band at 1402 cm −1 was found only for the apo state.
The COO − symmetric stretching region for the L-plastin headpiece in H 2 O solution also provides information regarding the coordination modes of the COO − groups to the metal ions despite other vibrational modes such as the CH 2 bending mode contributing to this region 15,16 . The amplitude at 1425 cm −1 , which corresponds to the band at 1423 cm −1 in Fig. 2b(C), was stronger in the Ca 2+ -loaded state than in the apo and Mg 2+ -loaded states, which undoubtedly reflects the coordination modes of the COO − groups to the metal ions as pseudo-bridging and/or bidentate modes. It is noted that the band at 1587 cm −1 for the L-plastin headpiece in the Ca 2+ -loaded state in H 2 O solution may reflect the coordination modes of COO − groups to Ca 2+ in the pseudo-bridging mode because the corresponding band in D 2 O solution was stronger in the Ca 2+ -loaded state than in the apo state 18 .
We also refer to the amide-I region because the spectral profiles for the Ca 2+ -loaded state in D 2 O and H 2 O solution were, respectively, different from those obtained for the apo and Mg 2+ -loaded states. The main peak position for amide-I and amide-I' was the same among the apo, Mg 2+ -loaded and Ca 2+ -loaded states, but the bandwidth at the 1652 cm −1 band for the Ca 2+ -loaded state was clearly narrower than for the apo and Mg 2+ -loaded states (Fig. 3b). This spectral difference may reflect a conformational difference such as α-helix formation induced by Ca 2+ binding. The bands at approximately 1682 cm −1 and 1633 cm −1 are assigned to a β-sheet conformation according to the empirical assignment of proteins 19,20 . The band at 1633 cm −1 was thought to not be induced by Ca 2+ binding because a shoulder at 1633 cm −1 was also observed in the apo and Mg 2+ -loaded states and because the amplitude at approximately 1682 cm −1 was observed to be constant for these states. www.nature.com/scientificreports www.nature.com/scientificreports/ FtIR spectra for the t-plastin headpiece. Figure 4 depicts the second-derivative spectra for a T-plastin headpiece in the apo, Mg 2+ -loaded and Ca 2+ -loaded states in D 2 O and H 2 O solutions. Here, we left out the Attenuated total reflection (ATR) spectra for the T-plastin headpiece since the second-derivative spectra provide information regarding the coordination structure, as well as the conformational changes induced by M 2+ in detail, as described in the FTIR section for the L-plastin headpiece. In the COO − antisymmetric region, two bands at 1580 and 1564 cm −1 in the apo state, two bands at 1583 and 1563 cm −1 in the Mg 2+ -loaded state and three bands at 1582, 1564 and 1555 cm −1 in the Ca 2+ -loaded state were observed (Fig. 4a). The band at 1580 cm −1 in the apo state ( Fig. 4a(A)) showed a 5 cm −1 downshift from the ionic Asp (1585 cm −1 ) [14][15][16] . The band at 1555 cm −1 in the Ca 2+ -loaded state ( Fig. 4a(C)) was due to the side-chain COO − groups binding to Ca 2+ in the bidentate coordination mode 15,16 but was 4-cm −1 higher than the corresponding band (1551 cm −1 ) for the L-plastin headpiece. In the region of the COO − symmetric stretch, the bands at 1421 and 1400 cm −1 in D 2 O solution and the bands at approximately 1422 and 1401 cm −1 in H 2 O solution were observed.
The band at 1614 cm −1 was due to amide-I' rather than the COO − antisymmetric stretch (Fig. 4a) because the corresponding bands were observed at 1620 cm −1 in H 2 O solution (Fig. 4b), which moved together with the band at 1693 cm −1 . The bands at 1693 and 1620 cm −1 were probably due to intermolecular interactions such as intermolecular β-strand since this protein seemed to easily aggregate with a spectral profile similar to that observed for a denatured protein 19,20 . CD spectra for plastin headpieces. The effects of Mg 2+ -and Ca 2+ -binding in the headpieces were analyzed also by CD spectroscopy. The CD spectrum in each state showed two troughs around 208 and 222 nm (Fig. 5). For the L-plastin headpiece (Fig. 5(A)), both peaks at 208 nm and 222 nm in the CD spectra were shifted toward more negative values due to Ca 2+ -binding from apo and Mg 2+ -loaded states. On the other hand, the T-plastin headpiece showed a spectral change with an increasingly negative value only around the peak at 222 nm (Fig. 5(B)). These results suggest that the two headpieces are folded as a single polypeptide and are rich in α-helices. No change occurred due to the presence of Mg 2+ on either of the headpieces, whereas some changes occurred due to Ca 2+ -binding on both the peptides 9 . The secondary structural change induced by Ca 2+ -binding was greater in the L-plastin headpiece than in the T-plastin headpiece. Therefore, the increase in the α-helix content in the L-plastin headpiece due to Ca 2+ -binding should be larger than that in the T-plastin headpiece. This result was consistent with that data obtained using FTIR spectroscopy because these headpieces had a Ca 2+ affinity in common with EF-hand calcium binding sites and less affinity for Mg 2+ ; therefore, Mg 2+ does not induce a conformational change in the headpieces.
FtIR spectra for the synthetic peptide analogs of the Ca 2+ -binding sites. Figure 6 depicts the second-derivative spectra for 17-residue synthetic peptide analogs of the Ca 2+ -binding sites I and II of the Land T-plastins in the wet film under D 2 O vapor atmosphere because the absorbance of the peptide in solution was too weak to analyze the amide I' and COO − stretching bands in detail. The bands at 1549 and 1553 cm −1 were detected for the peptide analogs corresponding to the site I and II of L-plastin in the Ca 2+ -loaded state, www.nature.com/scientificreports www.nature.com/scientificreports/ respectively ( Fig. 6a(B,D)). Therefore, we confirmed that the COO − of Glu at the 12th position is bound to Ca 2+ in the bidentate coordination mode 15,16 . Meanwhile, the spectral profiles for the peptide analogs corresponding to the site I and II of T-plastin were quite different from those obtained for the T-plastin headpiece. Bands at 1561 and 1565 cm −1 were detected for the peptide analogs corresponding to sites I and II of T-plastin, respectively ( Fig. 6b(B,D)), while a band at 1555 cm −1 was observed for the T-plastin headpiece in the Ca 2+ -loaded state (Fig. 4a(C)). The bands at 1681 and 1625 cm −1 in the amide I' region ( Fig. 6b(B)) suggested that the peptide analogs aggregated in the Ca 2+ -loaded state and that this aggregation disturbed the affinity of them for Ca 2+ . The same profile was also observed for the peptide analog corresponding to site II of T-plastin in the apo state ( Fig. 6b(C)). Bands at 1681 and 1625 cm −1 were not observed for the Ca 2+ -loaded state in Fig. 6b(D), which suggested that Ca 2+ reduced the aggregation of the peptide. We attempted to reduce the aggregation of site II of T-plastin by substitution of amino acid residue. At the present stage, we were not able to obtain information regarding the Ca 2+ -coordination structure for site II of T-plastin. However, as for site I of plastin T, we found that the mutant 17-residue peptide (C9K), where the cysteine was substituted for lysine at the 9 th position, did not aggregate. The FTIR spectra for the C9K peptide showed a band at 1560 cm −1 in the Ca 2+ -loaded state (data not shown), suggesting that the COO − group of Glu at the 12 th position is bound to Ca 2+ in the mode of pseudo-bridging coordination rather than in the mode of bidentate coordination.  www.nature.com/scientificreports www.nature.com/scientificreports/

Conclusions
The results obtained using the synthetic peptide analogs suggested that the lower sensitivity to Ca 2+ in the T-plastin headpiece may be related to the susceptibility to aggregation for the two Ca 2+ -binding sites. ATR-FTIR spectroscopy in combination with the use of a synthetic peptide analog approach is promising for understanding the correlation of Ca 2+ -binding coordination and the aggregation of Ca 2+ binding proteins.

Materials and Methods
sample preparation for plastin headpieces. Hexahistidine (His 6 )-tagged L-and T-plastin headpieces (L-plastin Δ1-100 and T-plastin Δ1-103) were expressed using a modified pET28a vector harboring their DNA sequences and Escherichia coli BL21(DE3) according to a previous report 9 . The expressed proteins were purified using Ni-NTA agarose (Qiagen). After cleaving the His 6 -tag on the resin with AcTEV protease, the eluted plastin headpieces were treated with trichloroacetic acid to remove contaminating Ca 2+ ions 21 . Further purification was performed by anion-exchange chromatography and size-exclusion chromatography with Resource-Q and Superdex 75 columns (GE Healthcare), respectively. sample preparation for the synthetic peptide analogs. We synthesized 17-residue peptide analogs corresponding to the two Ca 2+ -binding sites in the L-and T-plastins, as listed in Table 1, because the 17-residue peptide analogs for loop-helix F are the minimum number required for the Ca 2+ -binding property for site III of troponin C and site IV of akazara scallop troponin C 22,23 . The peptides were synthesized by the solid-phase method based on the Fmoc strategy 17,22,23 . A Fmoc-NH-SAL-PEG resin (Watanabe Chem.) solid-phase support containing the 4-[(2' ,4'-dimethoxyphenyl) N-Fmoc-aminomethyl]phenoxyacetamido group, named Rink-amide linker 24 , was used to provide the peptides with C-terminal amide. The peptide chain was constructed in a stepwise manner for respective amino acid residues. The coupling reaction of a side-chain protected Fmoc-amino acid was carried out with an equimolar reagent system of HBTU-HOBt in DMF containing a double equivalence of N-methyl morpholine. In every synthetic cycle, Fmoc-protecting groups of the elongating peptide chains were deblocked with mixed reagents of piperidine-DBU-HOBt, with concentrations of 8%(v/v), 2%(v/v), and 2.5%(w/v) in DMF, respectively. The additive HOBt was used to suppress the side reaction of the aspartic residue caused by piperidine 25 . After completion of elongation, the peptides were harvested by cleavage reaction with trifluoroacetic acid (TFA) containing EDT (4%), TIPS (6%), and water (2%). The crude peptides were dissolved in a LiCl (4%) solution of DMF and purified by reverse-phase HPLC. The molecular weights of the peptides were confirmed by MALDI-TOFMS with AXIMA (Shimadzu). The TFA carried-over from HPLC purification was completely removed with a size-exclusion column, PD-10 (GE Healthcare), in 0.1 M ammonium bicarbonate buffer solution (pH 8.5) containing 0.1 M KCl, because TFA causes disruptive overlapping of the FTIR signals 22,23 . Finally, the collected fractions of peptides were desalted with PD-10 in pure water.
FtIR measurements. Most of the experiments described for FTIR measuremrnts were performed in the same manner as in our previous works 17,26,27 . ATR-FTIR measurements were carried out for the L-and T-plastin headpieces at room temperature using a Perkin-Elmer Spectrum-One Fourier transform infrared spectrometer equipped with an ATR unit and an MCT detector with a resolution of 2 cm −1 27 . Interferograms from 200 scans were averaged for the series of measurements for L-plastin. On the other hand, interferograms from 500 scans were averaged for the series of measurements for the T-plastin headpiece since the sample concentration was lower (approximately half of the L-plastin headpiece concentration) due to partial aggregation 17 . Dry air gas was constantly pumped into the ATR unit of the spectrometer to suppress water vapor 17 . Approximately 10 μl of a sample solution was placed onto a Diamond/ZnSe 1-reflection top-plate (Perkin-Elmer). ATR-FTIR spectra for the solvents (buffer solutions) were measured in the same way. The treatment and analyses of the ATR-FTIR spectra have been described previously 17,27 . For the synthetic peptide analogs for the calcium binding sites of L-and T-plastins, ATR-FTIR measurements were also carried out for samples in a wet film under D 2 O vapor atmosphere to determine the absorbance intensity of the amide I' and COO − stretching modes 17 . CD measurements. CD spectra were measured using a spectrometer J-720 (Jasco) at room temperature.
The acquisition parameters were as follows: resolution, 0.2 nm; speed, 50 nm/min; response time, 2 s; bandwidth, 1 nm; and scan, 10. The 0.02 mM protein solution was prepared in 10 mM MOPS-KOH (pH 6.8), 100 mM KCl, and 0.05 mM EDTA for the M 2+ -free state and the same composition containing 2 mM MCl 2 , and for the M 2+ -loaded state. Each spectrum was subtracted with that from the buffer.