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Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy have been used to quantify metal binding to the phosphoprotein extractable from demineralized rat incisor dentine. Paramagnetic cation probes enable identification of the metal binding sites. Cations are able to diffuse across the protein surface while forming a relatively long-lived metal-phosphoprotein complex. The ability of the protein to sequester surface-mobile Ca(II) is discussed in terms of its ability to act as a possible nucleation site for the initial localization of Ca(II) within the dentine matrix. [ABSTRACT FROM AUTHOR]

The interaction between troponin-I and actin that underlies thin-filament regulation in striated muscle has been studied using proton magnetic resonance spectroscopy. A restricted portion of skeletal muscle troponin-I (residues 96-116) has previously been shown to be capable of inhibiting the MgATPase activity of actomyosin in a manner enhanced by tropomyosin [Syska et al. (1976) Biochem. J. 153, 375-387]. On the basis of homologous spectral effects for signals of specific groups observed in different complexes formed using the native proteins and a variety of defined peptides, it is concluded that the segment of troponin-I which has inhibitory activity interacts with the N-terminal region of actin, The surface of contact of the inhibitory segment of troponin-I with actin involves two regions of the N-terminal of actin. These are located between residues 1-7 and 19-44. The data are discussed in the context of a structural mechanism for the inhibition of myosin ATPase activation. [ABSTRACT FROM AUTHOR]

Reports on an analysis of [sup 1]hydrogen-nuclear magnetic resonance spectroscopic techniques of the conformation of the nitrogen terminal segment of the LC1 alkali light chain of rabbit skeletal muscle which has shown that this portion of the molecule adopts a well-defined elongated configuration. Finding that the rod-like feature is a consequence of the Ala/Pro-rich composition and discussion of the functional aspects of such conformational preference in this and similar segments in other proteins.

1HNMR is used to study the solution structure o f vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein binds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially, Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein. [ABSTRACT FROM AUTHOR]

Proton magnetic resonance spectroscopy has been used to study the cation(Mg2+ , Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the biding reaction. Complex formation with troponin I and the drug intercative conformational response to cation coordination and the binding of the drug and troponin I are discussed. [ABSTRACT FROM AUTHOR]

Identical tripeptides of the sequence X-Pro-Lys, where X is an unknown blocking group, were isolated from trypsin digests of bovine cardiac alkali light chain and the LC2 light chain of rabbit fast muscle. Chemical, electrophoretic and ¹H-NMR evidence characterized X as an unusual amino acid, α-N-trimethylalanine (Me3Ala), which was earlier reported as the N-terminal amino acid of the A1 alkali light chain of rabbit fast muscle [Henry et al. (1982) FEBS Lett. 144, 11–15]. The narrow Line width and chemical shift position (δ = 3.23 ppm) of the -N+-(CH3) protons of Me3Ala made ¹H-NMR spectroscopy a convenient method to search for this residue in other light chains. A survey of many different light chains showed that tiffs signal was present in all vertebrate striated muscle light chains of the A1-type (LC1, ‘essential’ light chains) and LC2-type (‘DTNB’-light chains, ‘phosphorylatable’ light chains) but was absent from all invertebrate muscle and vertebrate smooth muscle light chains tested. It was also absent from the vertebrate fast-muscle-specific A2-type (LC3) light chains. The spectral characteristics of these signals were consistent with their having arisen from the protons of an -N+-(CH3)3 grouping. Since no ε-trimethyllysine could be detected in acid hydrolysates of these proteins, it appears that Me3Ala is a general feature as the N-terminal amino acid in these light chains. ¹H-NMR studies on bovine cardiac myosin subfragment 1 (S1) showed that the Me3Ala methyl proton signal was clearly visible and that the spectrum more closely resembled that of a rabbit S1 isoenzyme, S1(A1), than S1(A2), suggesting that the 40-residue N-terminal segment of the alkali light chain in cardiac S1 also possesses a high segmental mobility. Addition of actin caused the same gross changes to the cardiac S1 spectrum as noted earlier for rabbit S1(A1) [Prince et al. (1981) Eur. J. Biochem. 121, 213–219]. In particular, a marked reduction in the segmental mobility of the N-terminal region of the alkali light chain was noted, consistent with a direct interaction of this area with actin. [ABSTRACT FROM AUTHOR]

Using assignments of resonances in the 1H NMR spectrum of calmodulin obtained by the use of large tryptic fragments of the molecule [Dalgarno, D. C., Klevit, R. E., Levine, B. A., Williams, R. J. P., Dobrowolski, Z., and Drabikowski, W. (1984) Eur. J. Biochem. 138, 281–289], the spectral changes which occur on Ca2+ binding to calmodulin have been examined in detail. Ca2+ binding occurs in two stages: the first two Ca2+ ions bind at sites III and IV (numbered from the N terminus) and the second two Ca2+ ions bind at sites I and II. The high-affinity binding causes perturbations of residues in both halves of the molecule, whereas binding at the two N-terminal sites only affects sidechains in that half of the molecule. The effects of binding Cd2+ to the Ca2+-binding sites of calmodulin have also been studied by 1H NMR. The cation induces spectral changes which are very similar to those seen for Ca2+, but some important differences do exist. [ABSTRACT FROM AUTHOR]

Two tryptic fragments of the Ca2+-binding protein calmodulin have been studied by high-resolution 1H NMR. TR1C (residues 1 – 77) spans the first two domains of the protein and TR2C (residues 78 – 148) spans the second two domains. The spectra indicate that each of the two-domain peptide assumes a conformation which is very close to that in the native protein. This characteristic holds both in the presence and in the absence of Ca2+ ions. Therefore, the resonance assignments obtained for the relatively simpler fragment spectra can be used to assign the spectrum of whole calmodulin. Analysis of the chemical shift patterns and nuclear Overhauser enhancement effects of several assigned resonances indicates that each half of calmodulin can be modeled after the two EF-Hand Ca2+-binding proteins for which crystal structures are available, namely parvalbumin and intestinal Ca2+ -binding protein. [ABSTRACT FROM AUTHOR]

High-resolution proton NMR spectrocopy has been used to study the solution structures of the subfragment 1 (S1) isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra are unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted in solution. These observations are in agreement with previous studies of the mixed isoenzymes [Highsmith et al. (1979) Biochemistry, 18, 4238 -4243]. Peptide amide exchange studies show also that the S 1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale,of the 1H-NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility as well as domains of different sidechain mobility. Close comparison of the S1(A1) and S1(A2) spectra indicate that the N-terminal 41 residues of the Al light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [S1(A1)-S1(A2)] obtained closely resembles the spectral simulation of the 41 -residue segment. Upon addition of actin, many of the narrow S1 resonances decrease in intensity or progressively disappear altogether, indicative of intermediate-slow exchange conditions consistent with the recognised high affinity between the two proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1 as has been suggested in earlier 1H-NMR studies referred to above. In particular, the differences noted between S 1(A 1) and S 1(A2) have now largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N-terminal region of the light chain in S 1(A 1). Together with other affinity chromatography results [Winstanley and Trayer (1979) Biochem. Soc. Trans. 7, 703-704], this is good evidence for a direct interaction between this area of S1(A1) and actin. [ABSTRACT FROM AUTHOR]

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations > 0.1 mM. ¹H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1-10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+-lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA ≈ 30 l/mol was calculated at lanthanide concentrations > 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the ¹H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cationlecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of stearic hindrance due to the extended conformation of the lecithin polar group. [ABSTRACT FROM AUTHOR]

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121,213–219]. It is now established that this N-terminal region of the A l-light chain interacts directly with the C-terminal region of actin in the acto-Sl (A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+ (CH3)3 protons of α-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11 — 15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1 (A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3A1a residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (<1.5 rim) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head. [ABSTRACT FROM AUTHOR]