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1.
Many of the structural domains involved in Ca2+ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that
differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the
development of novel, selective antagonists. The characteristics of Ca2+ current (ICa) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth
muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could
be represented by two time constants. Increasing ICa by increasing [Ca2+]o or with more negative holding potentials decreased both time constants. With Sr2+, Ba2+, or Na+ as the charge carrier, ICa inactivation was also represented by two time constants, both of which were larger than those found with Ca2+. With Ca2+, Sr2+, or Ba2+ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na+ (140 mM) was the charge carrier, voltages for Imax (−20 mV) or τmin (o mV) did not correspond. SSIN of ICa had a half-maximum voltage of −32±4 mV for Ca2+, −43 mV±5 mV for Sr2+, −41±5 mV for Ba2+, and −68±6 mV for Na+. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca2+, 6.8±0.3 for Sr2+, and 6.6±0.2 for Ba2+, representing four equivalent charges. When Na+ or Li+ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For ICa in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca2+ and Na+. The rate of recovery of ICa from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest
that inactivation of ICa in PV myocytes possess an intrinsic voltage dependence that is modified by Ca2+. For RPV but not rLV ICa, the charge of the permeating ion confers the voltage-dependency of SSIN. 相似文献
2.
The interaction of proteins with hydroxyapatite. III. Mechanism 总被引:5,自引:0,他引:5
The mechanism of protein binding to and elution from hydroxyapatite (HA) has been established. Binding occurs both by nonspecific attraction between protein positive charges and HA and by specific complexing of protein carboxyls with calcium loci on the mineral. Elution can take place either as the result of the nonspecific ion screening of charges or by the specific displacement of protein groups from sites on the column with which they had complexed. 相似文献
3.
Lymphocyte plasma membranes bind 45Ca2+ with three affinity sites: KAl = 4.0 . 106 M?1, KA2 = 8.5 . 104 M?1 and KA3 = 4.2 . 102 M?1, and Ca2+ binding capacities are 0.10, 1.2 and 85 nmoles Ca2+/mg protein. In the presence of 15 μg/ml ConA the Ca2+ binding constants were KA1 = 4.6 . 106 M?1, KA2 = 4.4 . 104 M?1 and KA3 = 4.2 . 102 M?1. The Ca2+ binding capacity was increased by ConA, to 0.13, 2.4 and 91 nmoles/mg protein. The Ca2+ ATPase activity of lymphocyte membranes was increased by ConA from 1 to 2 μmol P/protein × h. The 45Ca2+ uptake was stimulated by ConA and PHA to about 16 %. 相似文献
4.
《生物化学与生物物理学报:疾病的分子基础》2001,1535(2):153-163
Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb2+ at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb2+ on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb2+ induces a similar structure in osteocalcin as Ca2+ but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb2+ to osteocalcin (Kd=0.085 μM) than Ca2+ (Kd=1.25 mM). The hydroxyapatite binding assays show that Pb2+ causes an increased adsorption to hydroxyapatite, similar to Ca2+, but at 2–3 orders of magnitude lower concentration. Low Pb2+ levels (1 μM) in addition to physiological Ca2+ levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca2+ alone. These results suggest a molecular mechanism of Pb2+ toxicity where low Pb2+ levels can inappropriately perturb Ca2+ regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb2+-intoxicated animals. 相似文献
5.
Ca2+ and Mg2+ content of cellulose fibres is of relevance for a wide range of applications e.g. textile processing, pulp/paper, food. Sorption of Ca2+ and Mg2+ ions were found on lyocell type regenerated cellulose fibres. Higher affinity was found for Ca2+ ions compared to Mg2+ ions. At pH 9, fibre saturation was observed at a calcium binding capacity of 18–20 mmol/kg. A carboxylic group content of 18 mmol COOH per kg fibre material was determined based on the Methylene Blue absorption. This indicates a 1:1 molar stoichiometry between the carboxylic groups present in the fibres and the bound Ca2+ ions. Thus it is proposed that the salt in fibre shows the general composition (Cell-O? Ca2+ X?), X? being an anion bound in the salt to achieve charge neutrality.The sorption of Ca2+ also can be demonstrated by complex formation with 1,2-dihydroxy-9,10-anthraquinone (alizarin) which forms a red-violet Ca2+-complex. Colour fixation thus can be used as an indicator for the Ca2+-ions bound in the fibre. 相似文献
6.
Ann VanhoorenKristien Vanhee Katrien NoyelleZsuzsa Majer Marcel JoniauIgnace Hanssens 《Biophysical journal》2002,82(1):407-417
Thermodynamic parameters for the unfolding of as well as for the binding of Ca2+ to goat α-lactalbumin (GLA) and bovine α-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25°C. Among the different parameters available, the heat capacity increments (ΔCp) offer the most direct information for the associated conformational changes of the protein variants. The ΔCp values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective ΔCp values for the binding of Ca2+ are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca2+-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca2+-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in ΔCp values for the binding of Ca2+ is presumably in part related to this difference in charge distribution. 相似文献
7.
Johannes D. Clausen Jens Peter Andersen 《The Journal of biological chemistry》2010,285(27):20780-20792
The roles of Ser72, Glu90, and Lys297 at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca2+-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca2+ concentration of phosphorylation by Pi (“Ca2+ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca2+ in mutants with alanine or leucine replacement of Glu90, whereas arginine replacement of Glu90 or Ser72 allowed E2P formation from Pi even at luminal Ca2+ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF3− complex to luminal Ca2+. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu90 or Ser72, because the positive charge of the arginine side chain mimics Ca2+ occupying a luminally exposed low affinity Ca2+ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca2+ exit pathway. Mutants with alanine or leucine replacement of Glu90 further displayed a marked slowing of the Ca2+ binding transition as well as slowing of the dissociation of Ca2+ from Ca2E1 back toward the cytoplasm, thus demonstrating that Glu90 is also critical for the function of the cytoplasmically exposed Ca2+ sites on the opposite side of the membrane relative to where Glu90 is located. 相似文献
8.
Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[3H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the Bmax value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl? increased the number of L-glutamate binding sites and the effect was enhanced by Ca2+; Ca2+ alone had no significant effect; (b) the Cl?/Ca2+ -sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na+ ion was biphasic: low concentration of Na+ (< 5 mM) decreased Cl?7Ca2+ -de-pendent L-glutamate binding sites, whereas at higher concentrations of Na+ the binding of glutamate was found to increase either in the presence or absence of Ca2+ and Cl?. In addition, the K+ ion (50 mM) was found to decrease the Na+-independent and Cl?/Ca2--independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na?-dependent and Cl?/Ca2+-independent L-glutamate binding by 93%; in the presence of Cl, ?/Ca2+, the K+ ion decreased the Na+-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na+-dependent L-glutamate binding sites assayed in the absence of Ca2+ and Cl?. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na?-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na+-dependent nor APB (or Cl?/Ca2+)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX-SPM or CTX-PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L-glutamate. Furthermore, binding of L-glutamate to CTX-SPM or CTX-PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX-PSD fraction with 0.5% deoxycholate, 1.0% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L-glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L-glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ-aminobutyric acid and flunitrazepam binding sites, of the Ca2+-dependent K+ channel, and of the voltage-dependent Ca2+ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site. 相似文献
9.
The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K
d, 16.5 nM) and BPTI (K
d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K
d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K
d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K
d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K
d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K
d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel. 相似文献
10.
Farideh Ghomashchi Gajendra S. Naika James G. Bollinger Ahmed Aloulou Matthias Lehr Christina C. Leslie Michael H. Gelb 《The Journal of biological chemistry》2010,285(46):36100-36111
The cytosolic (group IV) phospholipase A2 (cPLA2s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA2s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA2β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca2+ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca2+ on the interfacial binding of mouse cPLA2β and its C2 domain to vesicles. Ca2+-dependent binding of mouse cPLA2β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca2+-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA2δ also displays Ca2+- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A1, and phospholipase A2 activities of the full set of mammalian cPLA2s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA2δ stands out as having relatively high phospholipase A1 activity. We also tested the susceptibility of all cPLA2 family members to a panel of previously reported inhibitors of human cPLA2α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA2β. These in vitro studies help determine the regulation and function of the cPLA2 family members. 相似文献
11.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,931(1):68-77
Two soluble Ca2+-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca2+-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different Km values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca2+ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca2+. A millimolar concentration of Mg2+ is required in addition to a micromolar concentration Ca2+ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C. 相似文献
12.
《Chemical Speciation and Bioavailability》2013,25(2):41-52
AbstractThis paper examines the inorganic complexing capacity of seawater, where chloride and sulfate ions are present in high concentration, towards mono- di- and tri-organotin(IV) cations which show a different trend of acidity, depending on cation charges, and a corresponding tendency to hydrolysis. By considering hydrolytic species and chloride and sulphate complex formation, a basic inorganic speciation model of organotins in synthetic seawater (Na+, K+, Ca2+, Mg2+, Cl?, SO42?) has been built up. The model has been extended to also consider interactions of organotins with carbonate and fluoride ions, which are other important components of seawater. Because of the strength of hydrolysis processes, the main complexes formed are in general mixed hydroxo-species. No species are formed by organotin cations and/or their hydroxo-species with fluoride owing to their very low concentration in fluoride, in comparison to the other components of seawater. In order to simplify calculations and to establish a cumulative inorganic binding capacity for seawater, we applied a chemical complexation model, according to which the major inorganic components of seawater are considered as a single salt BA. 相似文献
13.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies. 相似文献
14.
The calcium requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding 总被引:1,自引:0,他引:1
A radioligand receptor assay was used to determine the effect of Ca+2 and other cations on the binding of D-Ser-(t-Bu)6-desGly10EA GnRH (Buserelin acetate, Hoechst) to the gonadotropin releasing hormone receptor in rat pituitary membrane. Specific binding was reduced in the presence of CaCl2, BaCl2, MnCl2, Co(CH3COO)2, MgCl2, NaCl, or LaCl3, although these salts have markedly different effects on receptor mediated gonadotropin release from intact cells. Consistent with this finding, compounds which chelate cations increased specific binding of the ligand. The decreased binding is explained solely by an effect on receptor affinity, which was reduced from 4 × 109 M?1 in the presence of 10 mM CaCl2. Total receptor number was unchanged. These data suggest that the requirement for Ca+2 in GnRH-stimulated LH release is not mediated through a specific action of this ion at the level of receptor binding. 相似文献
15.
Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies. 相似文献
16.
In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca2+ concentration. The binding of Ca2+ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τr increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg2+ by Ca2+ at these sites (τr increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca2+ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd. 相似文献
17.
The surface charge density (σ′0) and the binding constant of Ca++ with charged groups on the outer surface of the membrane (KCa) were calculated from experimentally determined values of the shift of the current-voltage characteristic curves of calcium currents in the membrane of rat spinal ganglion neurons: σ′0 = 0.15 ± 0.05 e/nm2 and KCa = 70 ± 10 liters/mole. Using a three-barrier model the energy profile of the calcium channel of the membrane of these neurons was calculated for Ca++, Ba++, Cd++, Mn++, Co++, and verapamil. The calcium current was shown to be determined mainly by the depth of the potential hole corresponding to the outer binding site of the calcium channel. It is concluded from the results that the outer binding site of the calcium channel contains only one carboxyl group. 相似文献
18.
Ca2+ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca2+ binding in the NH2-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca2+ slowed HDX up to 2 orders of magnitude within the NH2-lobe and the NH2-lobe-associated TnI switch helix, implying that Ca2+ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca2+ and actin. Ca2+-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca2+ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca2+-mediated regulation: the coiled-coil''s NH2-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca2+-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca2+, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca2+ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features. 相似文献
19.
《Journal of molecular biology》2022,434(23):167872
EF-hand Ca2+-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca2+. Upon binding Ca2+, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca2+-binding affinity of CaM and most S100s in the absence of target is weak (CaKD > 1 μM). However, upon effector protein binding, the Ca2+ affinity of these proteins increases via heterotropic allostery (CaKD < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca2+ homeostasis and (ii) strict maintenance of Ca2+-signaling within a narrow dynamic range of free Ca2+ ion concentrations, [Ca2+]free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca2+ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function. 相似文献
20.
García MC Farías JM Escamilla J Sánchez-Armass S Sánchez JA 《The Journal of membrane biology》1999,168(2):141-148
The effects of a long-term blockade of L-type Ca2+ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers.
Ca2+ currents (I
Ca) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of I
Ca increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca2+ channel blocker nifedipine or in Ca2+-free conditions. A similar incubation period with Cd2+, an inorganic blocker, produced a moderate increase of 20% in peak I
Ca. The maximum mobilized charge (Q
max) increased by 50% in fibers preincubated in Ca2+-free solutions or in the presence of Cd2+.
Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd2+ prior to the isolation of the microsomal fraction doubled the number of 3H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the
number of binding sites is consistent with the increase in the peak amplitude of I
Ca as well as with the increase in Q
max.
Received: 31 August 1998/Revised: 7 December 1998 相似文献