The journal of physical chemistry. B, Jan 19, 2014
Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have ... more Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have become an extremely interesting class of molecules, especially for their potential biomedical applications. In this matter, it seemed important to define the nature and estimate the strength of their interaction with polynucleotides by different ways. We report in this work a systematic investigation through isothermal titration calorimetry of the thermodynamics of the association and dissociation of adenine and thymine derivatives, not previously performed. Then we use the results obtained on these simple systems as a basis for comparison with the binding of phospholipids functionalized with adenosine and thymidine to polyadenylic or polyuridylic acids applying the same experimental technique.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2015
Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine recept... more Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca(2+) channel complex that mediates Ca(2+) efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation-contraction coupling and defective CaM-RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca(2+)-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca(2+)-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca(2+)-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [(3)H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca(2+)-binding as well as defective interaction with RyR2.
Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitati... more Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation-contraction coupling. Defective CaM-RyR2 interaction is associated with heart failure. A novel CaM mutation (CaM(F90L)) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaM(F90L). F90L confers a deleterious effect on protein stability. Ca(2+)-binding studies reveal reduced Ca(2+)-binding affinity and a loss of co-operativity. Moreover, CaM(F90L) displays reduced RyR2 interaction and defective modulation of [(3)H]ryanodine binding. Hence, dysregulation of RyR2-mediated Ca(2+) release via aberrant CaM(F90L)-RyR2 interaction is a potential mechanism that underlies familial IVF.
Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multime... more Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2013
This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain... more This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.
Biochimica Et Biophysica Acta-proteins and Proteomics, 2010
The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRC... more The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRCA1/BARD1 complex demonstrates ubiquitin ligase activity and has been implicated in genomic stability and tumor suppression. Both proteins possess a structurally conserved C-terminal domain (BRCT). While BRCA1-BRCT has been shown to mediate BRCA1 interactions with phosphoproteins such as BRIP1 by recognizing the pSer-X-X-Phe motif, attempts to demonstrate analogous interactions of its dimeric counterpart BARD1-BRCT, have so far been unsuccessful. In this study, chemical-denaturation experiments of BARD1-BRCT domain suggest that its low thermodynamic stability (DeltaG=2.5 kcal/mol) at room temperature, may affect some of its biochemical properties, such as its interaction with phosphopeptides. The stability of BARD1-BRCT domain at 10 degrees C, increases to 7.5 kcal/mol and isothermal titration calorimetry (ITC) experiments at this lower temperature showed binding to the BRIP1 phosphopeptide via an enthalpy-driven interaction, which appears to be specific to the pSer-X-X-Phe peptide-binding motif. Substitution of either pSer at position 0 with Ser (non-phosphorylated peptide) or Phe with Val at position +3, leads to no-binding ITC results. While these findings are indicative that BRIP1 is a potential BARD1 binding partner, it becomes evident that in vitro binding assays involving the entire BARD1 protein and in vivo experiments are also needed to establish its binding partners and its potential role in tumor suppression pathways.
The anionic cyclodextrins (CDs), heptakis[6-(3-thiopropionate)-6-deoxy]-β-CD, heptakis[(6-(thio-e... more The anionic cyclodextrins (CDs), heptakis[6-(3-thiopropionate)-6-deoxy]-β-CD, heptakis[(6-(thio-ethanoate)-6-deoxy]-β-CD and partially phosphorylated 6-(aminoethylphosphate)-6-deoxy-β-CD as triethylammonium salts, bpsp.NHEt 3 , bpse.NHEt 3 and bphos.NHEt 3 , respectively, interacted with the positively charged picrate salt of heptakis[6-(guanidino)-6-deoxy]-β-CD, bguan.picrate, to form heterodimers as shown by NMR spectroscopic and microcalorimetric isothermal titration calorimetry (ITC) titrations in solution. Association constants, K a (M− 1) in DMSO-d 6 and in DMSO-d 6–H2O (80/20, v/v) were: bguan.picrate/bpsp.NHEt 3 , 6.4 × 105 and 5.9 × 104; bguan.picrate/bpse.NHEt 3 , 4.2 × 104 and 1.2 × 104; ITC data in DMSO agreed with those above: bguan.picrate/bpsp.NHEt 3 , K a = 4.7 ± 0.1 × 105 M− 1, ΔH = − 83.97 kJ mol− 1, ΔS = − 173.19 J mol− 1 K− 1; bguan.picrate/bpse.NHEt 3 , K a = 2.4 ± 0.2 × 105 M− 1, ΔH = − 88.49 kJ mol− 1 and ΔS = − 182.27 J mol− 1 K− 1. Heterodimers are highly stable in DMSO and less stable in DMSO–H2O. Multivalency in the interactions is manifested by positive cooperativity, negative enthalpy of formation (ΔH) and sizeable negative entropy (ΔS), in support of the development of well-ordered supramolecular structures in solution.
The journal of physical chemistry. B, Jan 19, 2014
Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have ... more Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have become an extremely interesting class of molecules, especially for their potential biomedical applications. In this matter, it seemed important to define the nature and estimate the strength of their interaction with polynucleotides by different ways. We report in this work a systematic investigation through isothermal titration calorimetry of the thermodynamics of the association and dissociation of adenine and thymine derivatives, not previously performed. Then we use the results obtained on these simple systems as a basis for comparison with the binding of phospholipids functionalized with adenosine and thymidine to polyadenylic or polyuridylic acids applying the same experimental technique.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2015
Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine recept... more Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca(2+) channel complex that mediates Ca(2+) efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation-contraction coupling and defective CaM-RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca(2+)-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca(2+)-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca(2+)-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [(3)H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca(2+)-binding as well as defective interaction with RyR2.
Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitati... more Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation-contraction coupling. Defective CaM-RyR2 interaction is associated with heart failure. A novel CaM mutation (CaM(F90L)) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaM(F90L). F90L confers a deleterious effect on protein stability. Ca(2+)-binding studies reveal reduced Ca(2+)-binding affinity and a loss of co-operativity. Moreover, CaM(F90L) displays reduced RyR2 interaction and defective modulation of [(3)H]ryanodine binding. Hence, dysregulation of RyR2-mediated Ca(2+) release via aberrant CaM(F90L)-RyR2 interaction is a potential mechanism that underlies familial IVF.
Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multime... more Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2013
This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain... more This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.
Biochimica Et Biophysica Acta-proteins and Proteomics, 2010
The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRC... more The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRCA1/BARD1 complex demonstrates ubiquitin ligase activity and has been implicated in genomic stability and tumor suppression. Both proteins possess a structurally conserved C-terminal domain (BRCT). While BRCA1-BRCT has been shown to mediate BRCA1 interactions with phosphoproteins such as BRIP1 by recognizing the pSer-X-X-Phe motif, attempts to demonstrate analogous interactions of its dimeric counterpart BARD1-BRCT, have so far been unsuccessful. In this study, chemical-denaturation experiments of BARD1-BRCT domain suggest that its low thermodynamic stability (DeltaG=2.5 kcal/mol) at room temperature, may affect some of its biochemical properties, such as its interaction with phosphopeptides. The stability of BARD1-BRCT domain at 10 degrees C, increases to 7.5 kcal/mol and isothermal titration calorimetry (ITC) experiments at this lower temperature showed binding to the BRIP1 phosphopeptide via an enthalpy-driven interaction, which appears to be specific to the pSer-X-X-Phe peptide-binding motif. Substitution of either pSer at position 0 with Ser (non-phosphorylated peptide) or Phe with Val at position +3, leads to no-binding ITC results. While these findings are indicative that BRIP1 is a potential BARD1 binding partner, it becomes evident that in vitro binding assays involving the entire BARD1 protein and in vivo experiments are also needed to establish its binding partners and its potential role in tumor suppression pathways.
The anionic cyclodextrins (CDs), heptakis[6-(3-thiopropionate)-6-deoxy]-β-CD, heptakis[(6-(thio-e... more The anionic cyclodextrins (CDs), heptakis[6-(3-thiopropionate)-6-deoxy]-β-CD, heptakis[(6-(thio-ethanoate)-6-deoxy]-β-CD and partially phosphorylated 6-(aminoethylphosphate)-6-deoxy-β-CD as triethylammonium salts, bpsp.NHEt 3 , bpse.NHEt 3 and bphos.NHEt 3 , respectively, interacted with the positively charged picrate salt of heptakis[6-(guanidino)-6-deoxy]-β-CD, bguan.picrate, to form heterodimers as shown by NMR spectroscopic and microcalorimetric isothermal titration calorimetry (ITC) titrations in solution. Association constants, K a (M− 1) in DMSO-d 6 and in DMSO-d 6–H2O (80/20, v/v) were: bguan.picrate/bpsp.NHEt 3 , 6.4 × 105 and 5.9 × 104; bguan.picrate/bpse.NHEt 3 , 4.2 × 104 and 1.2 × 104; ITC data in DMSO agreed with those above: bguan.picrate/bpsp.NHEt 3 , K a = 4.7 ± 0.1 × 105 M− 1, ΔH = − 83.97 kJ mol− 1, ΔS = − 173.19 J mol− 1 K− 1; bguan.picrate/bpse.NHEt 3 , K a = 2.4 ± 0.2 × 105 M− 1, ΔH = − 88.49 kJ mol− 1 and ΔS = − 182.27 J mol− 1 K− 1. Heterodimers are highly stable in DMSO and less stable in DMSO–H2O. Multivalency in the interactions is manifested by positive cooperativity, negative enthalpy of formation (ΔH) and sizeable negative entropy (ΔS), in support of the development of well-ordered supramolecular structures in solution.
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Papers by Angelos Thanassoulas