Neutralization of surface charges markedly affects the properties of bovine calbindin D9k.
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Amino Acids↗
Biomedical subjects
E Thulin
Publications and source records attributed to E Thulin.
Proline isomerism leads to multiple folded conformations of calbindin D9k: direct evidence from two-dimensional 1H NMR spectroscopy.
A complete analysis of calbindin D9k by two-dimensional 1H nuclear magnetic resonance spectroscopy has established the existence of two conformations for the folded protein in solution. Well-resolved major and minor resonances in a ratio of 3:1 are observed throughout the 1H NMR spectrum. Two-dimensional exchange experiments show that the major and minor species are related by an equilibrium process. Analysis of short proton-proton distances along the peptide backbone, identified by two-dimensional nuclear Overhauser effect spectroscopy, provides unambiguous evidence that the two forms of the folded protein differ only in the isomerization state of the peptide bond between Gly-42 and Pro-43. Cis-trans isomerism of Pro-43 is thereby directly identified as the cause of multiple conformations for the folded protein in solution. In addition, when Pro-43 is mutated to a glycine residue there is no indication of multiple conformations. These results provide evidence for the possibility of conformational heterogeneity in the native state of globular proteins.
Calbindins↗
Selective proton labelling of amino acids in deuterated bovine calbindin D9K. A way to simplify 1H-NMR spectra.
A technique for proton labelling of selected amino acids in deuterated calbindin D9K, heterologously expressed in E. coli, was developed in order to simplify and obtain higher resolution in 1H-NMR spectra. The spectra from two double-labelling experiments, Val plus Ser and Val plus Leu, when compared to the uniformly protonated protein showed a dramatically simpler pattern with low background signals and gave considerably sharper resonances due to reduced relaxation rates in the deuterated proteins. The selective proton labelling technique will enable detailed and rapid analysis of interesting domains of proteins and will also make the analysis of larger proteins feasible.
Amino Acids↗
Kinetics of calcium binding to calbindin mutants.
The kinetics of calcium dissociation from wild-type bovine calbindin D9k (the smallest protein known with a pair of EF-hand calcium-binding sites) and five mutants with single amino-acid substitutions and/or deletions has been studied by stopped-flow fluorescence methods, using the calcium chelator Quin 2. The modifications are confined to the N-terminal half of the molecule, at or near the first calcium-binding site (I). Substitutions and deletions of amino acids in the calcium-binding loop of site I primarily affect the rate of Ca2+ dissociation from this site with only minor effects on the dynamic properties of the C-terminal calcium-binding site (II). This finding corroborates and extends previous kinetic results obtained from 43Ca-NMR studies on the same set of mutants. By contrast, removal of the hydrogen bond between Tyr-13 and Glu-35, an interaction linking the two alpha-helices flanking site I, through replacement of Tyr-13 with Phe, has no observable effect on the rate of Ca2+ dissociation from the protein. Comparison of this kinetic data with binding-constant data, previously obtained in our laboratories, shows that the decrease in Ca2+-affinity of site I, observed in most mutants, is predominantly due to an increased off-rate from this site. At low ionic strengths the second-order rate constants for Ca2+-binding to both Ca2+ sites of calbindin D9k are calculated to be of the order of 10(9) M-1 s-1 for all proteins studied. At higher ionic strengths (0.1 M KCl) the rates of Ca2+ dissociation from both sites are increased by a factor of three or more, suggesting a transition state which is ionic in nature.
Amino Acids↗
The role of protein surface charges in ion binding.
Protein engineering is a means of probing the role of electrostatic interactions in protein functions; this elegant technique has been applied to the elucidation of electrostatic effects in enzyme catalysis. Here we show how the use of mutant proteins allows the determination of the contributions of individual charges to the free energy of ion binding to proteins. We have investigated the importance of three negatively charged side chains in the binding of Ca2+ to bovine calbindin D9K (ref.2): these are clustered around the calcium sites but are not directly involved as ligands. Each of these charges is found to contribute approximately 7 kJ mol-1 to the free energy of binding of two Ca2+ ions and to affect the cooperativity of Ca2+ binding. The influence of surface charges on ion binding to proteins may be more common than generally supposed and could have important consequences for protein function.
Calbindins↗
Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k.
The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.
Amino Acids↗
Purification of rabbit skeletal muscle troponin C.
Troponin C binds to phenyl-Sepharose in the presence of Ca2+ and can be eluted with EDTA. This property was used as an essential step in the purification of this protein from rabbit skeletal muscle. Troponin C was extracted with 6M urea from extensively washed ground muscle. The protein was bound to and eluted from DEAE-Sephadex, fractionated by size on Sephadex G75, and in a final step purified from UV-absorbing non-protein impurities on phenyl-Sepharose. The total yield of electrophoretically pure protein was 60 mg per 100 g of muscle, which is considerably higher than that previously obtained.
Animals↗
Structure-function relationships in EF-hand Ca2+-binding proteins. Protein engineering and biophysical studies of calbindin D9k.
Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.
Aminoquinolines↗
Is the binding of magnesium (II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance.
Previous reports on the interaction between calmodulin (CaM) and Mg2+ range from no binding to a binding constant of 10(4) M-1 [for a summary, see Cox, J. A., Comte, M., Malnoe, A., Berger, D., & Stein, E. A. (1984) Met. Ions Biol. Syst. 17, 215-273]. In order to resolve the controversy, we used 25Mg NMR to study the binding of Mg2+ to apo-CaM, CaM.Ca2(2)+ (in which sites III and IV are occupied by Ca2+), CaM.La2(3)+ (in which sites I and II are occupied by La3+), and the two tryptic fragments of calmodulin, TR1C (containing sites I and II of CaM) and TR2C (containing sites III and IV of CaM). In each system, a "titration set" and a "temperature set" were obtained, and the spectral data were analyzed by total band-shape analysis to calculate the association constant (Ka) and off-rate (koff). As in the case of Ca2+ binding, sites I and II and sites III and IV were treated as two sets of equivalent sites, and a Ca2+/Mg2+ competition experiment suggested that Mg2+ competes with Ca2+ for the same sites. For both CaM.Ca2(2)+ and TR1C, moderately large Ka (2000 and 3500 M-1, respectively) and moderate off-rates (koff = 2300 and 3000 s-1, respectively, at 25 degrees C) were observed. For both CaM.La2(3)+ and TR2C, binding of Mg2+ was weaker by a factor of ca. 10 (Ka = 300 and 200 M-1, respectively) while the off-rates were also moderate (koff = 3500 and 2200 s-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The binding of Ca2+, Mg2+ and Cd2+ to tryptic fragments of skeletal muscle troponin C. Cadmium-113 and proton NMR studies.
The structure of skeletal troponin C (sTnC) comprises two independently folded domains connected by an alpha-helical loop (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659). Both the NH2-terminal domain TR1C (9-84) and the COOH-terminal domain TR2C (89-159) can be obtained by limited tryptic cleavage. Here we report on proton and cadmium-113 NMR studies of Ca2+, Mg2+, and Cd2+ binding to these two separated domains. Our studies indicate that both halves retain a structure in the apo- and Ca2+-forms which resembles that of the intact protein. The events accompanying Ca2+ binding to these fragments are consistent with a biphasic binding pattern for sTnC, where the two sites in the COOH-terminal half are filled before those in the NH2-terminal half. Mg2+ only binds strongly to the two calcium-binding sites in the COOH-terminal half. The binding of this metal ion gives rise to a similar conformation for TR2C as that obtained with Ca2+. Whereas the binding of Ca2+ to the COOH-terminal part of sTnC takes place in a positive cooperative manner, the binding of this metal ion to TR2C occurs sequentially. Nevertheless, both Ca2+ ions bound to TR2C are in slow exchange (koff less than 10s-1). The binding of Mg24 both to sTnC and TR2C follows a sequential pattern with one site in slow exchange (koff less than 20 s-1) and one site in fast exchange (koff greater than 800 s-1). Binding of Cd2+ to TR2C occurs in a positive cooperative manner and results in the same conformation as observed with Ca2+. Cadmium-113 NMR spectra obtained at 5 degrees C confirm that two strong Cd2+-binding sites are localized in the COOH-terminal half and two weaker ones in the NH2-terminal half. It is concluded that these two proteolytic fragments comprise reasonable structural models for intact sTnC.
Animals↗
Evidence for the interaction between the calcium indicator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and calcium-binding proteins.
Stopped-flow and static difference spectroscopy experiments have shown that the calcium indicator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) interacts with several different calcium-binding proteins (beta-trypsin, parvalbumin, and calmodulin) and with serum albumin under experimental conditions commonly used in biophysical studies. The interaction decreases at high ionic strength. EDTA competes with BAPTA in the interaction with the proteins.
Animals↗
Expression of bovine intestinal calcium binding protein from a synthetic gene in Escherichia coli and characterization of the product.
Intestinal calcium binding proteins (ICaBP's) constitute a group of small vitamin D inducible proteins considered to play an important role in the absorption of dietary calcium. The mammalian ICaBP's are representatives of the "EF-hand" family of calcium binding proteins. As a first step in the application of protein engineering techniques to the study of structure-function relationships in mammalian ICaBP's, we have synthesized a gene encoding the minor A form (the native form lacking the two N-terminal amino acids) of bovine ICaBP employing a rapid, microscale gene synthesis technique based on "shotgun ligation" of sets of oligonucleotides. Expression of the synthetic gene from a plasmid containing the tac promoter in a lon protease deficient strain of Escherichia coli yielded the desired product at a level of about 1-2 wt % of total protein. During the purification of the ICaBP expressed in E. coli, a contaminant was strongly adhering to it but was efficiently removed by gel filtration after denaturation with urea. The minor A form of ICaBP produced in E. coli was characterized by its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by its total amino acid composition, partial amino acid sequence, UV absorption spectrum, and 360-MHz 1H NMR spectrum, showing beyond reasonable doubt its identity with the minor A form of ICaBP obtained from bovine intestines.
Amino Acid Sequence↗
Localization of a felodipine (dihydropyridine) binding site on calmodulin.
The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of CaM antagonist drugs through allosteric mechanisms [Mills, J.S., & Johnson, J.D. (1985) Biochemistry 24, 4897]. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to CAM and several of its proteolytic fragments. While TNS interacts with sites on both the amino-terminal half of the protein [proteolytic fragment TR1C (1-77)] and carboxy-terminal half [proteolytic fragment TR2C (78-148)], felodipine binding shows more selectivity. It binds in a Ca2+-dependent manner to the proteolytic fragments TM1 (1-106) and TR2E (1-90) but exhibits only weak affinity for TR1C (1-77) and TR2C (78-148). Furthermore, felodipine exhibits selectivity over TNS and trifluoperazine (TFP) in blocking the tryptic cleavage between residues 77 and 78. These studies indicate a selective binding of felodipine to a hydrophobic site existing in residues 1-90 and suggest that productive binding requires amino acids in the region 78-90. Although the felodipine binding site is preserved in fragment 1-106, the allosteric interactions between the prenylamine and the felodipine binding sites that are observed with intact CaM are not observed in this fragment. Rather, prenylamine simply displaces felodipine from its binding site on this fragment. Our results are consistent with calmodulin containing not less than two allosterically related hydrophobic drug binding sites. One of these sites (felodipine) appears to be localized in region 1-90 and the other one in region 78-148.
Animals↗
Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic, cadmium-113, and proton nuclear magnetic resonance studies.
Tryptic fragmentation of Ca2+-saturated calmodulin (CaM) takes place mainly at Lys-77; however, proteolysis can occur instead at Arg-74 or Lys-75. This cleavage pattern results in the production of three peptides each of the amino- and carboxy-terminal halves of CaM of slightly different length. A purification scheme for the three carboxy-terminal half-peptides is reported. Proton nuclear magnetic resonance (1H NMR) studies of peptides comprising the amino- or carboxy-terminal half of CaM reveal the great structural similarity between these two proteolytic fragments and the intact protein. Since this was observed for the apoprotein as well as the Ca2+-saturated protein, this means that the two halves of the protein are independently folded. A comparison of the changes in the 1H NMR spectra observed for the intact protein and the fragments upon addition of Ca2+ clearly identified sites III and IV as the two high-affinity binding sites. Furthermore, addition of Ca2+ or Cd2+ induces qualitatively similar changes in the spectra, thus indicating that Cd2+ is a reliable replacement for Ca2+ in these studies. Subsequent 113Cd NMR studies of trifluoperazine (TFP) binding to tryptic and thrombic fragments of calmodulin revealed the presence of two distinct drug binding sites, one located in the amino-terminal half and one located in the carboxy-terminal half. The spectral changes, induced upon addition of the antipsychotic drug, were similar to those observed upon binding of TFP to intact calmodulin. The strongest TFP binding site is located in the carboxy-terminal half.
Animals↗
Calcium and cadmium binding to troponin C. Evidence for cooperativity.
Proton NMR is used to compare the structural changes induced in bovine cardiac troponin C on binding of cadmium and calcium ions. The same spectral changes are observed for both ion species. The rate of the conformational changes associated with cadmium binding to the two high-affinity sites is slow, that associated with cadmium ions binding to the low-affinity site is high. 113Cd-NMR spectra of cardiac troponin C feature two signals interpreted as due to cadmium ions bound to the strong sites. Strong arguments are given in favour of cooperativity in binding of the first two cadmium or calcium ions to cardiac and skeletal muscle troponin C.
Allosteric Regulation↗
A 113Cd and 1H NMR study of the interaction of calmodulin with D600, trifluoperazine and some other hydrophobic drugs.
The interaction of calmodulin with D600 (a methoxy derivative of verapamil), trifluoperazine and some other drugs was studied by 113Cd and 1H NMR. All four cation binding sites of calmodulin were found to be affected by the binding of the drugs to calmodulin. The physiologically active and inactive forms of felodipine were found to give qualitatively the same changes in the 113Cd NMR spectra of calmodulin. The interpretation of this observation in terms of the physiological relevance of the binding to calmodulin is discussed. The binding constants for the two strongly bound trifluoperazine molecules were found to differ by one or two orders of magnitude. A competition study showed that trifluoperazine replaces D600 from at least one binding site on calmodulin. Moreover the binding of D600 was found to be calcium dependent. The data indicate that two calcium ions bound to calmodulin are sufficient to render the binding site(s) accessible for D600.
Animals↗
Cadmium-113 nuclear magnetic resonance studies of proteolytic fragments of calmodulin: assignment of strong and weak cation binding sites.
Proteolytic fragments of bovine testis calmodulin were obtained by limited proteolysis with trypsin or thrombin. Cadmium-113 NMR studies showed that the tryptic fragment encompassing Ca2+ binding domains III and IV (TR2C) gave rise to a spectrum identical with that of the native protein. Two thrombic fragments containing either domains I, II, and III [TM1-(1-106)] or the single domain IV [TM2-(107-148)] both gave rise to one broad resonance only. These data indicate that domains III and IV comprise the two high-affinity Ca2+ binding sites in intact calmodulin and that disturbance of the structural relationship between domain III and domain IV markedly reduces the affinity of these two sites for Ca2+ ions. These observations are discussed with respect to other published accounts concerning the sequence in which the four calcium domains in calmodulin are filled.
Animals↗
Characterization of the Ca2+ binding sites of calmodulin from bovine testis using 43Ca and 113Cd NMR.
The exchange rates of Ca2+ ions to the two classes of sites on calmodulin have been determined from the temperature dependence of the 43Ca NMR line width. The exchange rates were found to differ by a factor of about 40 at room temperature. The apparent pK value for one of these classes was estimated from the pH dependence of the 43Ca line width. The pK values of the two-high-affinity sites were found to differ about 0.1-1. Zn2+ ions were shown to bind to calmodulin and affect the exchange rates of Ca2+ and Cd2+ for all four sites.
Animals↗