Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

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.     

Effect of calcium and phosphatidic acid binding on the C2 domain of PKC alpha as studied by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.     

Conformational changes in concanavalin A associated with demetallization and alpha-methylmannose binding studied by Fourier transform infrared spectroscopy

Infrared spectra of concanavalin A have been obtained both in the absence and in the presence of the metal ions, Mn2+ and Ca2+, and the saccharide, alpha-methylmannose. Second derivative calculations have been used to determine the frequencies of the different amide I and II components. In the demetallized protein dissolved in H2O buffer, absorptions in the amide I, II and III regions at 1695 and 1634, 1532 and 1237 cm-1, respectively, are assigned to beta-structure, while absorptions at 1563 and both 1318 and 1343 cm-1 are assigned to turns and bends. After deuterium exchange, the residual amide II maximum in the difference spectrum shifts from 1538 to 1563 cm-1, indicating that exchange is faster in the beta-structure than in the turns. In the presence of Mn2+ and Ca2+, the amide II band component at 1532 cm-1 shifts 4-6 cm-1 to higher wavenumbers, and the amide I band component at 1634 shifts 1 cm-1 in the same direction, both in H2O and 2H2O buffers, suggesting changes in the hydrogen-bonding network of a large portion of the protein, particularly in the beta-sheet regions. The addition of alpha-methylmannose increases the magnitude of exchange from 55% to above 90%. Comparison with existing X-ray crystallographic data has been made, and the usefulness of FT-IR to complement this technique is discussed.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy.

Previous C13-NMR studies showed that two of the four internal aspartic acid residues (Asp-96 and Asp-115) of bacteriorhodopsin (bR) are protonated up to pH = 10, but no accurate pKa of these residues has been determined. In this work, infrared spectroscopy with the attenuated total reflection technique was used to characterize pH-dependent structural changes of ground-state, dark-adapted wild-type bacteriorhodopsin and its mutant (D96N) with aspartic acid-96 replaced by asparagine. Data indicated deprotonation of Asp-96 at high pH (pKa = 11.4 +/- 0.1), but no Asp-115 titration was observed. The analysis of the whole spectral region characteristic to complex conformational changes in the protein showed a more complicated titration with an additional pKa value (pKa1 = 9.3 +/- 0.3 and pKa2 = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pKa approximately 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.     

Conformational changes of the 120-kDa Na+/Ca2+ exchanger protein upon ligand binding: a Fourier transform infrared spectroscopy study

The 120-kDa Na+/Ca2+ exchanger was purified and reconstituted into lipid vesicles. The secondary structure composition of the exchanger was 39% alpha-helices, 20% beta-sheets, 25% beta-turns, and 16% random coils, as analyzed by Fourier transform infrared attenuated total reflection spectroscopy. The secondary structure composition of the COOH-terminal portion of the protein was compatible with a topology model containing 4-6 transmembrane segments. Furthermore, the secondary structure of the NH2-terminal portion of the cytoplasmic loop was analyzed and found to be different from that of the COOH-terminal portion. Ca2+ and/or the exchange inhibitory peptide (XIP) failed to affect the secondary structure of the 120-kDa protein. Tertiary structure modifications induced by Ca2+ and XIP were analyzed by monitoring the hydrogen/deuterium exchange rate for the reconstituted exchanger. In the absence of ligand, 51% of the protein was accessible to solvent. Ca2+ decreased accessibility to 40%, implicating the shielding of at least 103 amino acids. When both Ca2+ and XIP were added, accessibility increased to 66%. No modification was obtained when XIP was added alone. Likewise, in the presence of Ca2+, XIP failed to modify the tertiary structure of the 70-kDa protein, suggesting that XIP acts at the level of the COOH-terminal portion of the intracellular loop. The present data describe, for the first time, conformational changes of the Na+/Ca2+ exchanger induced by Ca2+ and XIP, compatible with an interaction model where regulatory Ca2+ and inhibitory XIP bind to distinct sites, and where XIP binding requires the presence of Ca2+.     

Coordination structures of Ca2+ and Mg2+ in Akazara scallop troponin C in solution. FTIR spectroscopy of side-chain COO- groups.

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 cm-1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm-1 in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm-1 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 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-I' 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.     

Phase behaviour and crystallinity of plant cuticular waxes studied by Fourier transform infrared spectroscopy

The phase behaviour of cuticular waxes from leaves of Hedera helix L. and Juglans regia L. was studied by Fourier transform infrared spectroscopy. For this purpose reconstituted waxes, isolated cuticular membranes, dewaxed polymer matrix membranes and whole leaves were studied in the horizontal attenuated total reflection and transmission modes. Melting curves of cuticular waxes were derived from temperature-dependent changes in the absorption maximum of the symmetric stretching mode of CH₂ groups (ν_s, at approx. 2856–2848 cm⁻¹). With increasing temperature absorption band doublets due to CH₂ scissoring (δ_sciss) and rocking (δ_rock) movements (at approx. 1473–1471 and 730–720 cm⁻¹, respectively) indicative of an orthorhombic arrangement of alkyl chains merged into a single peak. The area ratio of the peaks at approx. 720 and 730 cm⁻¹ was used as a measure for aliphatic crystallinity of plant cuticular waxes at a given temperature. The investigations of reconstituted cuticular waxes and those still embedded in isolated cuticles or in situ on the leaf produced comparable results. The findings are discussed in terms of the properties of the cuticular transport barrier. Received: 21 March 1997 / Accepted: 25 April 1997     

Ca2+-induced structural change in the Ca2+Mg2+ domain of troponin C detected by crosslinking

Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca²⁺. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca²⁺, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca²⁺, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the α-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the α-carbon distance His-125-Lys-136, do not exceed 14 Å. Comparison with the X-ray structure of troponin C (Herzberg, O, and James, M.N.G. (1985) Nature 313, 653–659) indicates that some of the above distances increase on Ca²⁺-binding.     

Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.     

Inhibition and partial reactions of Na,K-ATPase studied by Fourier transform infrared difference spectroscopy

Reaction-induced infrared (IR) difference spectroscopy with caged ATP and Na,K-ATPase allows us to differentiate unambiguously between phosphorylated and unphosphorylated states of the enzyme as well as of its ouabain complex. The IR spectral changes upon phosphoenzyme formation are characterized and interpreted. Our results show clearly that high Na(+) concentrations prevent the binding of ouabain with high affinity, which is consistent with the results of a corresponding kinetic study employing spectrofluorimetry and calorimetric titrations. This unexpected antagonism leading to low ouabain affinity is assumed related to a conformation of the protein, induced by low affinity binding of the third Na(+) ion. We thus conclude that not the free enzyme but a phosphorylated state of the reaction cycle preferentially binds ouabain and leads to the loss of hydrolytic activity.     

The effect of troponin C removal on the Ca2+-sensitive binding of Mg2+ AMPPNP to myofibrils

It was previously shown that when rabbit skeletal myofibrils are titrated with Mg2+ AMPPNP under conditions that result in the dissociation of cross-bridges from the thin filaments (i.e. 50% ethylene glycol, 0 degrees C), Ca2+-sensitive, biphasic binding is observed. These titrations have been repeated using myofibrils from which the troponin C has been selectively removed. The disappearance of both Ca2+ sensitivity and biphasic binding is taken as evidence that the Ca2+ sensitivity is due to Ca2+ binding to troponin C and the biphasic binding of Mg2+ AMPPNP observed in intact myofibrils is not due to packing constraints or steric hindrance.     

Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy

The second-derivative mode of the Fourier transform I.R. spectra of dried algal material has been applied to distinguish the carrageenans-producingStenogramme interrupta from the isomorphous speciesRhodymenia howeana. Spectra of the tetrasporophyteS. interrupta showed bands assigned to a -carrageenan type polysaccharide, while the gametophytic and cystocarpic plants showed the characteristic absorptions of -and -carrageenans. Results were confirmed by hot water extraction of samples of the three nuclear phases ofS. interrupta and characterization of the extracts by chemical analysis.Author for correspondence     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

TNP-AMP binding to the sarcoplasmic reticulum Ca(2+)-ATPase studied by infrared spectroscopy

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.