Báo cáo Y học: Coordination structures of Ca21 and Mg21 in Akazara scallop troponin C in solution FTIR spectroscopy of side-chain COO – groups

Eur. J. Biochem. 268, 6284–6290 (2001) q FEBS 2001 Coordination structures of Ca21 and Mg21 in Akazara scallop troponin C in solution FTIR spectroscopy of side-chain COO– groups Fumiaki Yumoto1, Masayuki Nara2, Hiroyuki Kagi3, Wakana Iwasaki1, Takao Ojima4, Kiyoyoshi Nishita4, Koji Nagata5 and Masaru Tanokura1 1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan; 2Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Chiba, Japan; 3Laboratory for Earthquake Chemistry, Graduate School of Science, University of Tokyo, Japan; 4Department of Marine Bioresources Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Japan; 5Biotechnology Research Center, University of Tokyo, Japan FTIR spectroscopy has been applied to study the coordination structures of Mg2‡ and Ca2‡ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2‡ binding site. The region of the COO– antisymmetric stretch provides information about the coordination modes of COO– groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm21 in the apo state, whereas additional bands were observed at 1543 and 1601 cm21 in the Ca2‡-bound and Mg2‡-bound states, respectively. The intensity of the band at 1567 cm21 in the Mg2‡-bound state was identical to that in the apo state. Therefore, the side-chain COO– group of Glu142 at the 12th position in the Ca2‡-binding site coordinates to Ca2‡ in the bidentate mode but does not interact with Mg2‡ directly. A slight upshift of COO– antisymmetric stretch due to Asp side-chains was also observed upon Mg2‡ and Ca2‡ binding. This indicates that the COO– groups of Asp131 and Asp133 interact with both Ca2‡ and Mg2‡ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the Muscle contraction of vertebrate skeletal and cardiac muscles is regulated by troponin in a Ca2‡ dependent manner [1]. Troponin contains three components, troponin C (TnC), troponin I, and troponin T, and TnC is the Ca2‡ binding component. In general, TnC contains two independent Ca2‡ binding domains, each of which consists of two EF-hand motifs [2]. Vertebrate TnCs bind three or four Ca2‡ ions in a molecule [3–5] and act as the Ca2‡ switch for muscle contraction associated with the binding and release of one or two Ca2‡ ions in the N-terminal domain. The N-terminal domain has therefore been called Correspondence to M. Tanokura, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan. Fax: ‡ 81 3 5841 8023, Tel.: ‡ 81 3 5841 5165, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp Abbreviations: CaM, calmodulin; M2+, divalent metal ion; TnC, troponin C. (Received 26 July 2001, revised 2 October 2001, accepted 8 October 2001) coordination structure of Mg2‡ was different from that of Ca2‡ in the Ca2‡-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2‡-bound and Ca2‡-bound states of Akazara scallop TnC, as spectral changes upon either Ca2‡ or Mg2‡ binding were very small in the infrared amide-I0 region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2‡ binding were larger than that upon Mg2‡ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC . Mg2‡-bound TnC . Ca2‡-bound TnC. These results suggest that the tertiary structures are different in the Ca2‡- and Mg2‡-bound states. The present study may provide direct evidence that the side-chain COO– groups in the Ca2‡-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC. Keywords: Mg2‡-ligation; Ca2‡-ligation; coordination structure; fluorescence spectroscopy. the regulatory domain and contains one or two low affinity Ca2‡-binding sites [6]. On the other hand, the C-terminal domain has been called the structural domain and contains two high-affinity sites. They also bind Mg2‡ and are called Ca2‡/Mg2‡ sites. Although the N-terminal low-affinity sites are called Ca2‡-specific sites, they also bind Mg2‡ weakly [7,8]. As intracellular Mg2‡ concentration is relatively high at about 1 mM [9,10], intracellular Mg2‡ ions are bound to the low-affinity Ca2‡-binding sites in addition to high-affinity sites in resting muscle cells [8]. It is therefore important to know the structural differences between Ca2‡-and Mg2‡-bound forms. In invertebrate muscles, troponin molecules and their TnCs bind less Ca2‡ than vertebrate TnCs, because they have lost the Ca2‡-binding ability at several sites due to the replacement of amino acids critical to chelate Ca2‡ [11]. Akazara scallop is one such invertebrate; its striated adductor muscle contains TnC that works as a Ca2‡ switch for contraction [12], and it binds only one Ca2‡ ion at the C-terminal EF-hand motif [13]. Akazara scallop TnC is therefore a curious and interesting molecule, as it regulates muscle contraction by binding a single Ca2‡ ion. q FEBS 2001 FTIR study of Akazara scallop TnC (Eur. J. Biochem. 268) 6285 room temperature at a flow rate of 0.5 mL·min21, as described previously [13,26]. The elution profile was monitored with a UV detector at 280 nm using AKTA-explorer (Amersham Pharmacia Biotech). The sample concentrations were 0.02 mM TnC, 150 mM KCl, 0.05 mM EDTA and 10 mM Mops/KOH (pH 6.8) for the apo state and 0.02 mM TnC, 120 mM KCl, 0.05 mM EDTA, 10 mM MgCl and 10 mM Mops/KOH (pH 6.8) for the Mg2‡-bound state. Fig. 1. Coordinating structures of the side-chain COO– groups to M21 in (A) unidentate, (B) bidentate, and (C) bridging modes. FTIR spectroscopy is a useful tool for examining protein conformations in an aqueous solution, in combi-nation with resolution-enhancement techniques such as second-derivative and Fourier-self-deconvolution methods [14,15]. A series of FTIR studies combined with these techniques have been performed to investigate the interactions between Ca2‡ and side-chain COO– groups in Ca2‡-binding proteins and have shown that the COO– stretching bands are useful for identifying the coordination structure of a side-chain COO– group interacting with Ca2‡ [16–20]. The COO– groups can coordinate to metal ions in three modes: unidentate, bidentate, and bridging modes (Fig. 1). A pseudo-bridging mode is a special case of the bridging mode, where one of the two ligands in the bridging coordination is replaced by a water molecule [21]. It is possible to characterize the coordination structures of the COO– groups to metal ions by the peak positions of COO– stretching modes [21,22]. A shift of COO– antisymmetric stretching vibration to a lower wavenumber is characteristic of the interaction between M2‡ and a COO– group in the bidentate coordination. On the other hand, the antisym-metric stretch of the COO– group not coordinated to M2‡ (ionic) or coordinated to M2‡ in either the unidentate mode or the pseudo-bridging mode shows a band in the range of 1605–1567 cm21. In the present study, we have measured FTIR spectra of Akazara scallop TnC to clarify the interaction of the side-chain COO– groups with Ca2‡ and Mg2‡ ions in the Ca2‡ binding site. The results have shown that the coordination structure of Mg2‡ is distinctly different from that of Ca2‡ in the Ca2‡ binding site; the Glu142 side-chain COO– group coordinates to Ca2‡ ion in bidentate but does not interact with Mg2‡ directly and that the Asp131 and Asp133 side-chain COO– groups coordinate in pseudo-bridging mode to both Mg2‡ and Ca2‡ ions. MATERIALS AND METHODS Materials Akazara scallop TnC was expressed and purified as described previously [23]. Apo protein was prepared by the previously described method [24]. Contaminating Ca2‡ was removed by treatment with trichloroacetic acid [25]. Gel-filtration experiments Gel-filtration experiments were performed on a SuperdexTM 75 HR 10/30 (Amersham Pharmacia Biotech) column at FTIR measurements To obtain reliable infrared spectra in the regions of the COO– antisymmetric stretch and the amide-I0 band, exchangeable protons in the protein were completely deuterated by incubating the apo protein dissolved in D2O at 60 8C for 60 min After cooling the solution to room temperature, the solution was freeze-dried. Apo samples for FTIR measurements were obtained by dissolving the powder of deuterated apo protein in D2O containing 40 mM Hepes/NaOD and 0.1 M KCl (pD 7.4). The samples of Mg2‡-bound proteins were obtained by dissolving the powder of deuterated apo protein in D2O containing 40 mM Hepes/NaOD and 0.1 M KCl in addition to 0.1 M MgCl2 or CaCl2 (pD 7.4). The samples prepared thus gave no NH proton signal in H-NMR spectra. The concentrations of sample solutions for FTIR measurements were 1.0 mM. A mixture of Mg2‡-bound TnC with Ca2‡ was prepared by mixing the Mg2‡-bound TnC solution mentioned above with a CaCl solution, and a mixture of Ca2‡-bound TnC with Mg2‡ by mixing the Ca2‡-bound TnC solution with a MgCl2 solution. The final concentrations were 25 mM MgCl2 and 50 mM CaCl2 for the former, and 50 mM MgCl2 and 25 mM CaCl2 for the latter. Both solutions contained 100 mM KCl and 0.5 mM TnC at pD 7.4. FTIR spectra were measured at room temperature on a PerkinElmer Spectrum-2000 Fourier-transform infrared spectrometer equipped with a TGS detector at 2 cm21 resolution. Interferograms from 200 scans were averaged to obtain one spectrum. Nitrogen gas was constantly pumped into the spectrometer to eliminate water vapor. About 0.012 mL of a sample solution was placed between two BaF2 plates separated by a 0.012-mm thick mylar spacer. The gap between the two BaF2 plates was sealed with aluminum tape to suppress the evaporationof water.Infrared spectra of the solvent (buffer solution) were measured in the same way. Data analyses for FTIR spectra To eliminate the contributions dueto D2Ofrom the spectrum of a protein solution, the spectrum of the solvent was subtracted from the spectrum of the protein solution after multiplying by an appropriate factor. Second derivative and difference spectra were calculated by using IGOR PRO 3.12 (WaveMetrics, Lake Oswego, USA) and the software supplied by PerkinElmer. The difference between Mg2‡-bound TnC and apo forms was carefully calculated by using the band at 1515 cm21 from to a tyrosine ring as an internal standard, because the band is not related to the Ca2‡ ligand. 6286 F. Yumoto et al. (Eur. J. Biochem. 268) q FEBS 2001 CD and fluorescence measurements CD spectra were acquired on a Jasco J-720 spectro-polarimeter at room temperature. All experiments were carried out as follows: 0.2 nm step resolution, 50 nm·min21 speed, 2 s response time, 1 nm bandwidth, and 10 scans. The sample concentrations were 0.02 mM TnC, 100 mM KCl, 0.05 mM EDTA and 10 mM Mops/KOH (pH 6.8) for the apo state and 0.02 mM TnC, 100 mM KCl, 0.05 mM EDTA, 2 mM MCl2 and 10 mM Mops/KOH (pH 6.8) for the apo state. The background signals from the buffer were subtracted from each spectrum. The a helical content of TnC was calculated with the program CONTIN [27]. Fluorescence was recorded on an RF-5300PC spectro-fluorimeter (Shimadzu) at room temperature. Fluorescence was excited at 295 nm. The sample concentrations were 0.005 mM TnC, 100 mM KCl, 0.05 mM EDTA and 10 mM Mops/KOH (pH 6.8) for the apo state and 0.005 mM TnC, 100 mM KCl, 0.05 mM EDTA, 10 mM MCl and 10 mM Mops/KOH (pH 6.8) for the M2‡-bound state. RESULTS FT-IR spectra for apo, Mg21-bound and Ca21-bound Akazara scallop TnC Figure 2 shows the infrared second-derivative spectra of apo, Mg2‡-bound and Ca2‡-bound Akazara scallop TnC in D O over the range of 1750–1350 cm21.The patterns of the amide-I0 region (1700–1620 cm21) and COO– stretching region (1430–1370 cm21) were similar to one another; the bands at 1674, 1646 and 1402–1 cm21 were observed in each state. The band at 1515 cm21 due to a tyrosine ring was almost the same among the three states. Significant differences were observed in the region of COO– antisymmetric stretch (1620–1530 cm21). In the apo state (Fig. 2A), two strong bands at 1584 and 1567 cm21 were observed, which were due to the side-chain COO– groups of Asp and Glu, respectively [16]. In addition to these, bands were observed at 1601 cm21 in the Mg2‡-bound state (Fig. 2B) and at 1543 cm21 in the Ca2‡-bound state (Fig. 2C). The intensity of the 1567-cm21 band in the Mg2‡-bound state was stronger than that in the Ca2‡-bound one, but identical with that in the apo one. The intensity of the 1584-cm21 band in the Mg2‡-bound state was obviously smaller than that in apo one, whereas the corresponding band in the Ca2‡-bound state showed a band at 1586 cm21, 2-cm21 higher in the wavenumber than that in the apo state. The weak bands at 1605 cm21 for apo state and at 1610 cm21 for the Ca2‡-bound state may also be due to side-chain COO– groups. Figure 3 shows the 1750–1350 cm21 region of the difference spectra between the M2‡-bound and the apo states. As can be predicted from the second-derivative spectra (Fig. 2), positive bands appeared in the COO– antisymmetric stretching region at 1602 cm21 for the Mg2‡-bound state, and the positive bands at 1613 and 1542 cm21 for the Ca2‡-bound state. The former corre-sponds to the band at 1601 cm21 and the latter corresponds to the 1610 and 1543 cm21 bands in Fig. 2. Furthermore, Fig. 3 provides additional significant information on the COO– antisymmetric stretching region; the negative band at 1581 cm21 in Fig. 3A, the positive one at 1592 cm21 and Fig. 2. FTIR second-derivative spectra of (A) apo (B) Mg21-bound, and (C) Ca21-bound Akazara scallop troponin C in solutions containing 40 mM Hepes-NaOD (pD 7.4) and 100 mM KCl. Second derivatives are multiplied by 21. the negative one at 1573 cm21 in Fig. 3B. In the amide-I0 region the intensity of 1646 cm21 increased slightly upon Ca2‡-binding, whereas such a change was not observed upon Mg2‡-binding. In addition, it should be noted that an upshift of COO– symmetric stretching band from 1396 to 1428–5 cm21 occurred upon Mg2‡ or Ca2‡ binding. Infrared measurements for TnC in the coexistence of Mg21 and Ca21 We furthermore examined FTIR spectra for a mixture of Mg2‡-bound TnC with Ca2‡ (Mg2‡/Ca2‡ ˆ 1 : 2) and a mixture of Ca2‡-bound TnC with Mg2‡ (Mg2‡/ Ca2‡ ˆ 2 : 1) (data not shown), in order to confirm that TnC interacts with Mg2‡ only in the Ca2‡-binding site. This is because the spectral change of COO– antisymmetric stretching band upon Mg2‡ binding was obviously larger than that upon Ca2‡ binding, as can be seen in Fig. 3. As a result, the spectra of these mixed systems showed a spectral pattern identical with the Ca2‡-bound state (Fig. 2C). It means that the Mg2‡ ion was replaced by the Ca2‡ ion in the Ca2‡-binding site but the Ca2‡ ion was not replaced by the Mg2‡ ion. It is therefore concluded that the difference in the COO– antisymmetric stretching regionmentioned above is obviously due to the side-chain COO– groups in the Ca2‡-binding site and not due to the other side-chain COO– q FEBS 2001 FTIR study of Akazara scallop TnC (Eur. J. Biochem. 268) 6287 Fig. 3. FTIR difference spectra of Akazara scallop troponin C induced by (A) Mg21 binding and (B) Ca21 binding. groups. We also confirmed that the intensity of the band at 1567 cm21 for the Mg2‡-bound TnC was stronger than that for the mixture of Mg2‡-bound TnC with Ca2‡, indicating that the band at 1543 cm21 stemmed from the band at 1567 cm21 for the Mg2‡-bound state. Re-examination of the infrared spectra for [EDTA–M]2– complex We re-examined the FTIR spectra for ethylene diamine tetra acetate ion (EDTA4–) complexing with Mg2‡ and Ca2‡ for the purpose of understanding the behavior of COO– antisymmetric stretching band. EDTA4– showed a COO– antisymmetric band at 1584 cm21 in the apo state as described in a previous study [18]. [EDTA–Mg]2– and [EDTA–Ca]2–, respectively, showed the most intense bands at 1601 and 1588 cm21 in the COO– antisymmetric stretching region at pH 8.0. These bands were very close to the bands at 1602 and 1591 cm21 observed for the difference spectra of TnC induced by Mg2‡ binding and Ca2‡ binding (Fig. 3), respectively. CD spectra of apo, Mg21-bound, and Ca21-bound TnC The effects of Mg2‡ and Ca2‡ binding in Akazara scallop TnC were also investigated by CD spectroscopy. The CD spectrum in each state showed two negative peaks at 208 and 222 nm (Fig. 4A). Very small changes occurred upon Fig. 4. CD spectra (A), gel-filtration chromatograms (B) and fluorescence spectra (C) of Akazara scallop troponin C in the apo (–), Mg21-bound (- - -), and Ca21-bound (······) states. Mg2‡ binding whereas some small changes occurred upon Ca2‡ binding. The a helix content for apo, Mg2‡-bound, and Ca2‡-bound TnC were estimated as 36, 41 and 39%, respectively, according to the method reported by Provencher et al. [27]. As a result, most of the secondary structures were conserved among them. Gel-filtration chromatography and fluorescence spectroscopy for apo, Mg21-bound, and Ca21-bound TnC The effects of Mg2‡ or Ca2‡ binding on Akazara scallop TnC were also examined by gel-filtration chromatography and fluorescence spectroscopy. The gel-filtration experi-ments showed that the elution volumes are different among apo, Mg2‡-bound and Ca2‡-bound TnCs (Fig. 4B). In the case of BSA as the reference sample, the elutionvolumewas not changed by Mg2‡ or Ca2‡ addition. As a result, the molecular sizes of TnC had the following order: apo TnC . Mg2‡-bound TnC . Ca2‡-bound TnC. The fluorescence spectra over the range of 290–450 nm showed that the intensities at 350 nm had the following order: 6288 F. Yumoto et al. (Eur. J. Biochem. 268) Ca2‡-bound TnC . Mg2‡-bound TnC < apo TnC (Fig. 4C). Consequently, the tertiary structure of Ca2‡-bound TnC was slightly different from that of Mg2‡-bound one as well as that of the apo state. DISCUSSION Our interest in the present study is focused on the coordination structures of Mg2‡ and Ca2‡ in the Ca2‡-binding site as well as the protein conformation. The Ca2‡ binding loop (site IV) of this protein is composed of DTDGSGTVDYEE (residues 131–142) [13]. Applying the general rule of EF-hand motif [28] to this protein, the COO– groups of Asp131, Asp133 and Glu142 should coordinate to Ca2‡ directly. On the basis of the crystal structure of vertebrate TnCs [29,30], the COO– group of Glu at the 12th position in site IV possibly coordinates to Ca2‡ ion in the bidentate mode and the COO– groups of Asp at the 1st and 3rd positions in the unidentate mode. The relationship between the coordination structures of the COO– group and the peak positions of COO– stretching bands in FTIR spectra has been elucidated for acetate compounds not only by experimental studies [31] but also by ab initio molecular orbital calculation [32]: (a) the peak position of the COO– antisymmetric stretch of the unidentate species is higher than that of the ionic species (free COO– group), which is in turn higher than that of the bidentate species; (b) the position of the pseudo-bridging species is close to or a little higher than that of the ionic species. Such trends apply to carboxylic acid compounds such as Asp, Glu, and EDTA. Moreover, we have reconfirmed that [EDTA–Mg]2– and [EDTA–Ca]2–, models for a pseudo-bridging mode, show the bands at 1601 and 1588 cm21 for COO– antisymmetric stretching vibration, respectively. We have interpreted the bands in the COO– antisym-metric stretching region for Akazara scallop TnC in relation to the coordination structures of the COO– group and the peak positions of COO– stretching bands mentioned above. (a) The 1543-cm21 band in Fig. 2C is due to side-chain Glu142 COO– coordinated to the Ca2‡ ion in the bidentate mode. This band was not observed in the Mg2‡-bound state and the intensity at 1567 cm21 in the Mg2‡-bound state was the same as that in the apo state. Therefore, this COO– group does not coordinate Mg2‡ directly. (b) The bands at 1602 cm21 for the Mg2‡-bound state in Fig. 3A and at 1592 cm21 for the Ca2‡-bound state in Fig. 3B indicate that the COO– groups of Asp131 and Asp133 interact with Ca2‡ as well as Mg2‡ in the pseudo-bridging mode. This is the direct evidence that Akazara scallop TnC interacts with Mg2‡ in the single Ca2‡-binding site. (c) The shift of COO– symmetric stretch from 1396 to 1425 cm21 in Fig. 3A also reflects that the COO– groups interact with Mg2‡ in the pseudo-bridging mode, because the peak positions for the COO– symmetric and antisymmetric stretching vibrations move together [19,21,22]. In addition, the weak band at 1605 cm21 for the apo state and 1610 cm21 for the Ca2‡-bound state may be due to side-chain COO– groups but cannot be assigned at the present state. It should be noted that these interpretations have been carried out, regardless of the crystal structures of EF-hand proteins reported so far. q FEBS 2001 We have found that there is a difference in the Ca2‡ ligation betweenAkazara scallop TnC andpike parvalbumin pI 4.10 [16]. The band position at 1543 cm21 due to the bidentate Glu142 COO– group of Akazara scallop TnC is different from that at 1553 cm21 due to the bidentate Glu COO– groups of pike parvalbumin pI 4.10 as well as bovine CaM [16,17], but is close to that at 1544 cm21 due to recoverin [20]. According to the ab initio molecular orbital calculation study[32], the band position of COO– stretching vibration is related to the changes in the OCO angle and CO length of the COO– group; the variation of 18 in OCO angle or 0.001 nm of the CO distance gives rise to an observable change of the band position of COO– stretching bands. Therefore, this difference suggests that the Ca2‡ coordi-nation structure of Akazara scallop TnC is similar to but not identical with those of parvalbumin and bovine CaM. We also have found that there is a difference in the Mg2‡ ligation; the side-chain COO– of Glu142 has shown a band at 1566 cm21 in the Mg2‡-bound state, whereas the corresponding COO– groups of Glu62 and Glu101 of pike parvalbumin pI 4.10 showed a band at 1584 cm21 [16]. This means that the COO– group of Glu142 of TnC does not interact with Mg2‡, while the COO– groups of Glu62 and Glu101 of parvalbumin seem to interact with Mg2‡ in the pseudo-bridging mode. The present study also provides more conclusive information about the side-chain COO– groups of Asp131 and Asp133 at the first and third positions than the previous studies on pike parvalbumin pI 4.10 and bovine CaM [16,17], as Akazara scallop TnC has a single Ca2‡ binding site. It should be noted that the spectral change caused by Mg2‡ binding is larger than that caused by Ca2‡ binding, as shown in Fig. 3. One possibility is that the Asp139 side-chain COO– group at the ninth position interacts with Mg2‡ in the pseudo-bridging mode, but does not interact with Ca2‡ directly. Another possibility is that the spectral change upon Ca2‡ binding is small in appearance because of the slight shift of COO– antisymmetric stretching vibration. Most of the secondary structures of Akazara scallop TnC seem to be maintained among the apo, Mg2‡-bound and Ca2‡-bound states according to FTIR and CD spectra. The present results show that Akazara scallop TnC has less a helix content than vertebrate TnC. The spectral pattern of amide-I0 is consistent with the result obtained by CD spectroscopy, because the main amide-I0 band at 1646 cm21 is too low for a protein with a high a helix content. It is, however, notable that such a spectral pattern has also been observed in other EF-hand Ca2‡-binding proteins such as pike parvalbumin pI 4.10, bovine CaM and vertebrate TnC [14,15,33,34]. The spectral changes in Fig. 3 seem smaller than those of vertebrate TnC and CaM reported by Trewhella et al. [33]. This is reasonable because the Akazara scallop TnC contains only a single active Ca2‡-binding site in a molecule, whereas the bovine CaM and vertebrate TnC contain four active Ca2‡-binding sites. It is interesting that the band corresponding to marker I (< 1658 cm21) observed in Ca2‡-bound bovine CaM [17] and bullfrog TnC (F. Yumoto, M. Nara and M. Tamokura, unpublished data) in D O was not observed in the second-derivative spectra of Ca2‡-bound Akazara scallop TnC (Fig. 2C). Unlike bovine CaM and vertebrate TnC [33], most of the secondary structures of Akazara scallop TnC may not be affected upon either Mg2‡ or Ca2‡ binding. ... - tailieumienphi.vn