Fourier transform infrared spectroscopic studies of Ca(2+)-binding proteins

The secondary structures of calmodulin and parvalbumin are well established from X-ray diffraction and nuclear magnetic resonance spectroscopic studies, which indicate that these proteins are predominantly alpha-helical in character. Recent infrared studies have nevertheless suggested that the helical structures present in these proteins in solution are not the standard alpha-helix but rather some kind of distorted helices [Trewhella, J., et al. (1989) Biochemistry 28, 1294]. The evidence for this was the unusually low amide I frequency for calmodulin and troponin C in 2H2O solution. The studies presented here, however, suggest that the helical structures in these proteins are not significantly distorted, for two reasons. First, distorted helical structures have weaker hydrogen bonds than the standard alpha-helix and would therefore be expected to absorb at a higher rather than a lower frequency. Second, distorted helical structures would absorb at an unusual frequency in H2O solutions which is not the case for the proteins studied here. The band frequency of these proteins is observed to occur at a frequency observed with other proteins known to contain predominantly alpha-helical structures. Quantitative analysis of the FT-IR spectra of calmodulin (67% alpha-helix) and parvalbumin (68% alpha-helix) in H2O in the presence of Ca2+ gives helical contents similar to those reported by X-ray studies. This raises the question as to why these proteins in H2O show a normal frequency for the presence of alpha-helical structures and an abnormal frequency in 2H2O. Addition of deuterated glycerol to the proteins in 2H2O solutions results in a significant shift of absorbance to higher frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oriented secondary structure in integral membrane proteins. I. Circular dichroism and infrared spectroscopy of cytochrome oxidase in multilamellar films.

The circular dichroism (CD) of cytochrome oxidase in solution indicates the presence of both alpha-helix (approximately 37%) and B-sheet (approximately 18%). In oriented films generated by the isopotential spin-dry method, the CD measured normal to the film shows a marked decrease in the negative bands at 222 and 208 nm, and a decrease and red shift in the positive band near 195 nm, relative to solution spectra. These features are characteristic of alpha-helices oriented with their helix axes along the direction of light propagation. A quantitative estimate of the orientation, based on the ratio of the rotational strengths of the 208-nm band in the film and in solution, leads to an average angle between the helix axis and the normal to the film, phi alpha of approximately 39 degrees. A method for analyzing infrared (IR) linear dichroism is developed that can be applied to proteins with comparable amounts of alpha-helix and beta-sheet. From analysis of the amide I band, phi alpha is found to lie between 20 and 36 degrees, depending on the angle that the amide I transition moment forms with the helix axis. A survey of the literature on the amide I transition moment direction indicates that a value of approximately 27 degrees is appropriate for standard alpha-helical systems, such as those in cytochrome oxidase. A larger value, near 40 degrees, is reasonable for systems that have distorted alpha-helices, as evidenced by amide I frequencies above 1,660 cm-1, as is the case of bacteriorhodopsin. This conclusion supports phi alpha approximately 36 degrees from IR linear dichroism, in agreement with the CD results. Linear dichroism in the amide I and amide II region indicates that the beta-sheet in cytochrome oxidase is oriented with the carbonyl groups nearly parallel to the plane of the membrane and the chain direction inclined at approximately 40 degrees to the normal. Comparison of these results with tentative identification of transmembrane helices from sequence data suggests that either some of the transmembrane helices are inclined at an unexpectedly large angle to the normal, or the number of such helices has been overestimated. Some putative transmembrane helices may be beta-strands spanning the membrane.     

Ca2+ binding sites in calmodulin and troponin C alter interhelical angle movements

Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism.     

Interaction of calmodulin with troponin I and the troponin-tropomyosin-actin complex. Effect of Ca2+ and Sr2+ ions.

In an effort to elucidate the mechanism of calmodulin regulation of muscle contraction, we investigated the interaction between calmodulin and troponin components in the presence of Ca2+ or Sr2+ by the use of ultracentrifugation methods and polyacrylamide-gel electrophoresis. Skeletal-muscle troponin C bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was absent, calmodulin bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was present, calmodulin hardly bound to troponin I even in the presence of bivalent cations. Trifluoperazine, a calmodulin antagonist, inhibited the bivalent-cation-dependent interaction between calmodulin and troponin I. Calmodulin migrated more slowly in the presence of Sr2+ than it did in the presence of EGTA but faster than it did in the presence of Ca2+ on polyacrylamide-gel electrophoresis under non-denaturing conditions. It is concluded that troponin T is not required in the calmodulin regulation of muscle contraction because troponin T inhibits the bivalent-cation-dependent interaction between calmodulin and troponin I and because calmodulin binds to troponin I and dissociates it from the tropomyosin-actin complex in a bivalent-cation-dependent manner. Sr2+-induced exposure of the hydrophobic region enables calmodulin to bind to troponin I, as is the case with Ca2+.     

FTIRS in H2O demonstrates that collagen monomers undergo a conformational transition prior to thermal self-assembly in vitro

The assembly of type I collagen molecules into native fibrils can be accomplished in vitro in solutions at physiological ionic strength and pH by raising the temperature above 30 degrees C. The thermal self-assembly reaction exhibits a distinct lag phase. This lag phase has been proposed to be evidence for a conformational transition in the monomer. Fourier transform infrared spectroscopy (FTIRS) is a very sensitive probe of the H-bonded states within the triple helix. The carbonyl group spectrum (amide I, 1700-1600 cm-1) has been investigated in collagen/H2O solutions at 1 mg/mL under self-assembly conditions from 4 to 34 degrees C and, in the same range, at a higher ionic strength where self-assembly does not occur. The deconvoluted spectra show three very clear bands at approximately 1660, 1644, and 1630 cm-1. These bands vary in both frequency maxima and relative intensity over the temperature range examined. Spectra were also obtained in the amide II and III regions. Spectral changes were evident in the 22-26 degrees C range, under fibril-forming conditions, which lead to the hypothesis that the triple helix of the semiflexible collagen molecule is actually perfected during the lag phase, facilitating nucleation and intermolecular interaction. Further spectral changes after fibrils do form show that the molecules are once again distorted as they are bent to fit within the fibrils.     

A laser Raman spectroscopic study of Ca2+ binding to troponin C.

Laser Raman spectroscopy has been used detect structural changes in troponin C induced by Ca2+ binding. Addition of Ca2+ - Mg2+ sites produces perturbations in the amide III region of the spectrum indicative of increased alpha-helical content, and in regions of the spectrum corresponding to carboxylate, thiol, and phenol side chains. However, Ca2+ binding to the low affinity Ca2+ - specific sites is not detected by laser Raman spectral changes.     

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.     

Model for the interaction of amphiphilic helices with troponin C and calmodulin

Crystals of troponin C are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).     

Construction and molecular dynamics simulation of calmodulin in the extended and in a bent conformation.

Analysis of sequence similarity and comparison of the three-dimensional (3D) structures of troponin C and calmodulin have revealed a sequence in the central helix of calmodulin with a high probability for bending. The three amino acids known to form a bend in the N-terminal portion of troponin C are also found in the central helix of calmodulin. The modelling of a bent calmodulin structure, using the dihedral angles of the three residues in the bend of troponin C as a 3D template, results in a conformation of calmodulin where the N- and C-terminal domains are able to form contacts. Dynamics simulations starting from the X-ray structure of calmodulin and from the modelled bent calmodulin were carried out to compare flexibility and correlated movements of Ca2+ in the binding loops. Both conformations of calmodulin remained stable during the period of simulation. In the simulation of calmodulin in the extended form, the motions of the Ca2+ atoms in the two domains (Ca2+1 and Ca2+2 in one domain, and Ca2+3 and Ca2+4 in the other) are correlated. In the simulation of the bent form, an additional correlation between the Ca atoms in the two different domains is observed. The results are compatible with the occurrence of a bent conformation of calmodulin in the presence of targets, and with increased Ca2+ affinity and cooperativity of the Ca(2+)-binding loops in the calmodulin-peptide complexes.     

Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.     

Laser Raman characterization of conformational changes in sarcoplasmic reticulum induced by temperature, Ca2+, and Mg2+

Laser Raman spectroscopy has been used to examine the conformations of the protein and phospholipid components of sarcoplasmic reticulum from rabbit white skeletal muscle. The phospholipid component is shown to have the conformation of fluid, liquid-crystalline lipids, even at 10 degrees C, and no breaks in the lipid conformation are observed in the range of 10-37 degrees C. Protein (predominantly the Ca2+-dependent ATPase) conformation is shown to contain very little beta-sheet structure under all conditions. Absolute content of alpha-helix and random coil or beta-turn could not be determined because of interference in the amide I and III regions. However, the Ca2+-ATPase in sarcoplasmic reticulum appears to undergo a conformational change at 15-18 degrees C which involves removal of a portion of the tryptophan residues from an aqueous environment and an increase in alpha-helical content. This conformation change coincides with a change in slope of Arrhenius plots of ATP hydrolysis activity. Increasing concentrations of Ca2+ and Mg2+ appear to slightly decrease the alpha-helical content of sarcoplasmic reticulum protein.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.     

Ca2+ in contractile processes

The concept of Ca2+ regulation, first discovered and developed in muscle research, is historically surveyed. Ca2+ regulation mechanisms in actomyosin-dependent contractile processes are compared, emphasis being placed on the great diversity. The mode of action of Ca2+ is discussed with the examples of troponin and calmodulin, the most differentiated and conservative Ca2+-receptor proteins, respectively.     

Analysis of the regulatory and structural defects of troponin C central helix mutants.

Five deletion mutants of the D/E linker region of the troponin C central helix were tested for conformational and functional differences from wild-type troponin C. The mutants were in the region 87KEDAKGKSEEE97: dEDA, dKG, dKGK, dKEDAKGK, and dSEEE, designed to change the length of the central helix and the orientation of the Ca(2+)-binding domains relative to each other [Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710]. Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other. All mutants undergo Ca(2+)-dependent conformational changes as detected by changes in electrophoretic mobility, alpha-helix content, and hydrophobic exposure. Deletions of the central helix do not extensively alter the thermal stability of troponin C, as determined by temperature-dependent loss of alpha-helix. There are differences among the mutants that do not correlate with function. All troponin C mutants show Ca(2+)-dependent interaction with troponin I and T in polyacrylamide gels. Troponin I-troponin C interaction was also analyzed by Ca(2+)-dependent increase in the monomer/excimer ratio of tropinin I and relief of inhibition of the actomyosin S1 ATPase. While all mutants retain basic function, dKGK, dKEDAKGK, and dEDA have altered interaction with troponin I in the absence of Ca2+. dSEEE differs in conformation from wild type, but it is normal in functional assays. This conserved region of the D/E linker is not required for interaction with troponin I in the presence or absence of urea.     

Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins

The Ca2+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils; Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of the CDTA-treated myofibrils.     

Secondary structure of the membrane-bound form of the pore-forming domain of colicin A. An attenuated total-reflection polarized Fourier-transform infrared spectroscopy study.

The structure of the pore-forming domain of the bacterial toxin colicin A was studied by attenuated total-reflection polarized Fourier-transform infrared spectroscopy. This channel-forming fragment interacts with dimyristoylglycerophosphoglycerol (Myr2GroPGro) vesicles and forms disk-like complexes. Analysis of the shape of the amide I' band indicates that its secondary structure is not affected by the pH 5.0-7.2. However, 5-10% of the peptide amino acids adopt an alpha-helical structure upon complex formation with Myr2GroPGro, while the random-coil and beta-sheet structure contents decrease. Interestingly, the increase in alpha-helical content is essentially due to an increase in the high-frequency component of the alpha-helical domain of amide I'. The fact that only this component was 90 degrees polarized (i.e. the helix is parallel to the acyl chain) suggests that only this particular type of helix is associated with the Myr2GroPGro bilayer.     

The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels

We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B). These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.     

Study of calcium binding to calmodulin using laser raman spectroscopy

Laser Raman spectroscopy has been used to study calcium binding to calmodulin, Ca2+-dependent regulator protein. Cation binding accompanied by spectral changes of tyrosine residues in the regions of Fermi-resonance doublet and 1600-1620 cm-1, of some carboxylate-containing residues, amide I, III and C-C(N) skeletal vibrations. Amide III contour analysis and calculations of Amide I contours show that complexation causes peptide backbone conformational changes characterized mainly by increased alpha-helical content.     

The cilia of Paramecium tetraurelia contain both Ca2+-dependent and Ca2+-inhibitable calmodulin-binding proteins.

To identify protein targets for calmodulin (CaM) in the cilia of Paramecium tetraurelia, we employed a 125I-CaM blot assay after resolution of ciliary proteins on SDS/polyacrylamide gels. Two distinct types of CaM-binding proteins were detected. One group bound 125I-CaM at free Ca2+ concentrations above 0.5-1 microM and included a major binding activity of 63 kDa (C63) and activities of 126 kDa (C126), 96 kDa (C96), and 36 kDa (C36). CaM bound these proteins with high (nanomolar) affinity and specificity relative to related Ca2+ receptors. The second type of protein bound 125I-CaM only when the free Ca2+ concentration was below 1-2 microM and included polypeptides of 95 kDa (E95) and 105 kDa (E105). E105 may also contain Ca2+-dependent binding sites for CaM. Both E95 and E105 exhibited strong specificity for Paramecium CaM over bovine CaM. Ciliary subfractionation experiments suggested that C63, C126, C96, E95, and E105 are bound to the axoneme, whereas C36 is a soluble and/or membrane-associated protein. Additional Ca2+-dependent CaM-binding proteins of 63, 70, and 120 kDa were found associated with ciliary membrane vesicles. In support of these results, filtration binding assays also indicated high-affinity binding sites for CaM on isolated intact axonemes and suggested the presence of both Ca2+-dependent and Ca2+-inhibitable targets. Like E95 and E105, the Ca2+-inhibitable CaM-binding sites showed strong preference for Paramecium CaM over vertebrate CaM and troponin C. Together, these results suggest that CaM has multiple targets in the cilium and hence may regulate ciliary motility in a complex and pleiotropic fashion.