RESUMO
Quantitative NMR spectroscopy is a useful and important tool for analysis of various mixtures. Recently, in addition of traditional quantitative 1D (1)H and (13)C NMR methods, a variety of pulse sequences aimed for quantitative or semiquantitative analysis have been developed. To obtain actual usable results from quantitative spectra, they must be processed and analyzed with suitable software. Currently, there are many processing packages available from spectrometer manufacturers and third party developers, and most of them are capable of analyzing and integration of quantitative spectra. However, they are mainly aimed for processing single or few spectra, and are slow and difficult to use when large numbers of spectra and signals are being analyzed, even when using pre-saved integration areas or custom scripting features. In this article, we present a novel software, ImatraNMR, designed for batch analysis of quantitative spectra. In addition to capability of analyzing large number of spectra, it provides results in text and CSV formats, allowing further data-analysis using spreadsheet programs or general analysis programs, such as Matlab. The software is written with Java, and thus it should run in any platform capable of providing Java Runtime Environment version 1.6 or newer, however, currently it has only been tested with Windows and Linux (Ubuntu 10.04). The software is free for non-commercial use, and is provided with source code upon request.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Software , Algoritmos , Glicemia/análise , Humanos , Imageamento por Ressonância Magnética , Óleos/análise , Plasma/química , Análise de Componente PrincipalRESUMO
Quantitative NMR spectroscopy is a useful tool for the analysis of various mixtures. Usually (1)H NMR is used for quantitative measurements, but in many cases the better signal dispersion offered by (13)C NMR is beneficial. However, the low natural abundance of (13)C and long T(1) relaxation times make the acquisition of quantitative (13)C spectra with adequate signal-to-noise ratio time-consuming. The use of polarization transfer experiments such as DEPT or INEPT can offer improved signal intensity and faster repetition rate, but yield non-quantitative results. In this paper we present a pulse sequence based on constant-time INEPT, Q-INEPT-CT, which is capable of producing quantitative carbon spectra with better sensitivity and/or in less time than traditional quantitative (13)C. Additionally, the constant length of the sequence means that signal loss due to relaxation effects can be relatively easily corrected. Thus, the presented sequence is a valuable tool when quantitative carbon data is required quickly and/or low-concentration samples are involved.
Assuntos
Algoritmos , Carbono/análise , Carbono/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Isótopos de Carbono/química , Simulação por ComputadorRESUMO
An improvement of the GBIRD-filter is presented. The current approach utilizes Carr-Purcell-Meiboom-Gill type pulse train during the BIRD delay. The method enables recording of purely absorptive 1D spectrum using only one isotope editing element. In the current method, the parent signal leakage due to JHH evolution during the BIRD delay is considerably smaller than in the conventional approach. As a consequence, the t1-noise is smaller also in 2D applications, such as GBIRD-filtered HSQC.
RESUMO
The interaction between troponin C (TnC) and troponin I (TnI) is essential for the regulation of muscle contraction. There are several binding sites for TnI on TnC that are differentially occupied depending on the phase of the contraction/relaxation cycle. TnI and TnC interact in an antiparallel fashion with each other. The C-domain of cTnC and the N-domain region of cTnI(residues 33-70) always interact under physiological conditions, whereas the interaction between regulatory regions of TnC and TnI (residues 128-166) is calcium dependent. Previously, it has been shown that levosimendan, a calcium sensitizer used as a treatment for acute heart failure, can interact with both domains of isolated cTnC. To understand which interaction is relevant for the mechanism of calcium sensitization, we used a more complete troponin model obtained by complexing cTnI(32-79) and cTnI(128-180) with calcium-saturated cTnC(CS). The cTnI peptides bound to cTnC(CS) to form a 1:1:1 complex. The interaction of levosimendan with this complex was followed by 1H-(15)N heteronuclear correlation spectroscopy. It was clear that based on chemical shift changes, cTnI(32-79) blocked the levosimendan interaction sites on the C-domain, whereas cTnI(128-180) did not compete with levosimendan for the binding site on the N-domain. Hence, the effective binding site of levosimendan on cTnC resulting in the calcium-sensitizing effect is located in the regulatory domain (N-domain).
Assuntos
Cardiotônicos/metabolismo , Hidrazonas/metabolismo , Fragmentos de Peptídeos/metabolismo , Piridazinas/metabolismo , Troponina C/metabolismo , Troponina I/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Cisteína/metabolismo , Interações Medicamentosas , Humanos , Modelos Moleculares , Contração Miocárdica , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Simendana , Trítio , Troponina C/química , Troponina C/genética , Troponina I/metabolismoRESUMO
A three-dimensional approach for measuring 15N relaxation times is described. Instead of selecting particular values for the relaxation period, in the proposed method the relaxation period is incremented periodically in order to create a 3D spectrum. This additional frequency domain of the transformed spectrum contains the relaxation time information in the T1 and T2 linewidths, and thus the longitudinal and transverse 15N relaxation times can be measured without determination of 2D cross peak volumes/intensities and subsequent curve fitting procedures.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Ubiquitinas/química , Análise dos Mínimos Quadrados , Isótopos de Nitrogênio , Processamento de Sinais Assistido por ComputadorRESUMO
A fluorinated analogue of coniferyl alcohol has been reported to be a specific inhibitor of oxidases involved in the biosynthesis of lignin. The Z isomer of beta-fluoro-coniferyl alcohol was synthesized and used for the preparation of dehydrogenation polymers (DHPs) and was also tested on lignin producing suspension cultures of spruce (Picea abies (L.) Karst.). The growth of the cells or the production of lignin by the suspension cultures was not significantly affected by the addition of fluoroconiferyl alcohol. This analogue did not form polymers quite as easily as did coniferyl alcohol in oxidation with hydrogen peroxide and horseradish peroxidase. In both cases the beta-fluoroconiferyl alcohol became incorporated in the polymeric product. We were unable to detect any specific inhibition of peroxidase activity, which is at variance with earlier reports of pronounced inhibition of lignin biosynthesis in poplar plantlets by fluoroconiferin, a potential inhibitor of oxidases involved in lignin biosynthesis.
Assuntos
Lignina/antagonistas & inibidores , Fenóis/isolamento & purificação , Picea/metabolismo , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Cinética , Lignina/biossíntese , Fenóis/química , Picea/citologiaAssuntos
Domínio Catalítico , Proteínas de Choque Térmico/química , Isomerases/química , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Radioisótopos de Carbono/química , Humanos , Isoenzimas/química , Radioisótopos de Nitrogênio/química , Isomerases de Dissulfetos de Proteínas , PrótonsRESUMO
Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca(2+) and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca(2+)-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca(2+)-saturated states.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/metabolismo , Motivos EF Hand , Saccharopolyspora/química , Proteínas de Bactérias , Proteínas de Ligação ao Cálcio/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Saccharopolyspora/metabolismo , TitulometriaRESUMO
Two methods for the measurement of (2)J(NCalpha) and (1)J(NCalpha) in (15)N/(13)C-labeled small and medium-size proteins are described. The current approach is based on simplified (13)C(alpha)-coupled (15)N HSQC spectra, where the two (2)J(NCalpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NCalpha) doublets between the two subspectra provides an accurate value for (1)J(NCalpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Humanos , Ubiquitinas/químicaRESUMO
Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca(2+)-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca(2+)-loaded regulatory domain of recombinant cTnC(C35S) was observed. The changes in the NMR spectra of the N-domain of full-length cTnC(C35S), due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnC(A-Cys), where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnC(C35S) and cTnC(A-Cys). The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca(2+)-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN(3)), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.
Assuntos
Cálcio/metabolismo , Hidrazonas/metabolismo , Miocárdio/metabolismo , Piridazinas/metabolismo , Troponina C/metabolismo , Espectroscopia de Ressonância Magnética , Ligação Proteica , Simendana , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Conformations of the regulatory domain of cardiac troponin C (cNTnC) were studied by means of residual dipolar couplings measured from samples dissolved in dilute liquid crystals. Changes in the main chain HN residual dipolar couplings revealed a conformational change in cNTnC due to the complexation with the second binding region (amino acids 148-163) of cardiac troponin I (cTnI). Formation of the complex is accompanied with a molecular realignment in the liquid crystal. The residual dipolar couplings measured for apo-cNTnC and the complex with TnI were in agreement with the values computed from the corresponding closed and open solution structures, whereas for the calcium-loaded conformation the correlation and quality factor were only modest. Ca2+-cNTnC may be subject to conformational exchange. The data support the model that cardiac troponin C functions as a calcium-dependent open-closed switch, such as the skeletal troponin C.
Assuntos
Troponina C/química , Troponina I/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Modelos Moleculares , Músculo Esquelético , Miocárdio , Conformação Proteica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Transverse relaxation-optimized NMR experiment (TROSY) for the measurement of three-bond scalar coupling constant between (1)H(alpha)(i-1) and (15)N(i) defining the dihedral angle psi is described. The triple-spin-state-selective experiment allows measurement of (3)J(H(alpha)N) from (13)C(alpha), (15)N, and (1)H(N) correlation spectra H(2)O with minimum resonance overlap. Transverse relaxation of (13)C(alpha) spin is minimized by using spin-state-selective filtering and by acquiring a signal longer in (15)N-dimension in a manner of semi-constant-time TROSY evolution. The (3)J(H(alpha))(N) values obtained with the proposed alpha/beta-HN(CO)CA-J TROSY scheme are in good agreement with the values measured earlier from ubiquitin in D(2)O using the HCACO[N] experiment.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Algoritmos , Ubiquitinas/químicaRESUMO
The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.
Assuntos
Fusão de Membrana , Capuzes de RNA , Sequência de Aminoácidos , Membrana Celular/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Vírus da Floresta de Semliki/enzimologia , Homologia de Sequência de Aminoácidos , Triptofano/químicaRESUMO
Fractionation of proteins from perinatal rat brain was monitored using a neurite outgrowth assay. Two neurite-promoting proteins, HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin) and amphoterin, were isolated, cloned and produced by baculovirus expression for structural and functional studies. HB-GAM is highly expressed in embryonic and early post-natal fiber pathways of the nervous system, and it enhances axonal growth/guidance by binding to N-syndecan (syndecan-3) at the neuron surface. N-syndecan in turn communicates with the cytoskeleton through the cortactin/src-kinase pathway to enhance neurite extension. In addition to N-syndecan, the chondroitin sulfate proteoglycan RPTP beta/zeta (receptor-type tyrosine phosphatase beta/zeta) is implicated in the receptor mechanism of HB-GAM. HB-GAM is also prominently expressed in developing and regenerating bone as a matrix-bound cue for migration of osteoblasts/osteoblast precursors to the site of bone deposition. HB-GAM is suggested to regulate motility in osteoblasts through a similar mechanism as in neurons. Structural studies using heteronuclear NMR reveal two similar protein domains in HB-GAM, both consisting of three anti-parallel beta-strands. Search of sequence databases shows that the beta structures of HB-GAM and of the similar domains of MK (midkine) correspond to the thrombospondin type I (TSR) sequence motif. We suggest that the TSR sequence motif, found in several neurite outgrowth-promoting and other cell surface and matrix-binding proteins, defines a beta structure similar to those found in HB-GAM and MK. In general, amphoterin is highly expressed in immature and transformed cells. We suggest a model, according to which amphoterin is an autocrine/paracrine regulator of invasive migration. Amphoterin binds to RAGE (receptor of advanced glycation end products), an immunoglubulin superfamily member related to N-CAM (neural cell adhesion molecule), that communicates with the GTPases Cdc42 and Rac to regulate cell motility. In addition, ligation of RAGE by amphoterin activates NF-kappaB to regulate transcription.
Assuntos
Proteínas de Transporte/fisiologia , Movimento Celular/fisiologia , Citocinas/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , Citocinas/genética , Citocinas/isolamento & purificação , Proteína HMGB1 , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/isolamento & purificação , Modelos Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Neuritos/fisiologia , Neurônios/fisiologia , Ratos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/fisiologiaRESUMO
Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycin-resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N-terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids.
Assuntos
Antibacterianos/farmacologia , Peptídeos/farmacologia , Proteínas/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos , Dicroísmo Circular , Resistência Microbiana a Medicamentos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Modelos Moleculares , Peptídeos/química , Peptídeos/isolamento & purificação , Conformação Proteica , Proteínas/química , Proteínas/isolamento & purificação , Rana temporaria , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Resistência a VancomicinaRESUMO
The NH tautomerism of five Mg-free chlorophyll a and b derivatives 2-6 was studied utilizing NMR spectroscopy and molecular modeling. The results from the dynamic NMR measurements of the chlorins revealed that substituent effects contribute crucially to the free energy of activation (DeltaG(double dagger)) in the NH tautomeric processes. An intermediate tautomer for the total tautomeric NH exchange in a chlorin was observed for the first time, when the (1)H NMR spectra of chlorin e(6) TME (3) and rhodin g(7) TME (4) (TME = trimethyl ester) were measured at lower temperatures. The lower energy barriers (DeltaG(1)(double dagger)) obtained for the formation of the intermediate tautomers of 3 and 4, assigned to the N(22)-H, N(24)-H trans-tautomer, were 10.8 and 10.6 kcal/mol, respectively. The energy barrier (DeltaG(2)(double dagger) value) for the total tautomeric NH exchange in the five chlorins was found to vary from 13.6 kcal/mol to values higher than 18 kcal/mol. The lowest DeltaG(2)(double dagger) value (13.6 kcal/mol) was obtained for rhodochlorin XV dimethyl ester (2), which was the only chlorophyll derivative lacking the C(15) substituent. In the case of chlorins 4 and 5, the steric crowding around the methoxycarbonylmethyl group at C(15) raised the DeltaG(2)(double dagger) activation free-energy to 17.1 kcal/mol. However, the highest energy barrier with DeltaG(2)(double dagger) > 18 kcal/mol was observed for the NH exchange of pyropheophorbide a methyl ester (6), possessing the macrocycle rigidifying isocyclic ring E. Our results demonstrate that the steric strain, arising either from the steric crowding around the bulky substituent at C(15) or the macrocycle rigidifying isocyclic ring E, slows down the NH tautomeric process. We suggest that deformations in the chlorin skeleton are closely connected to the NH tautomeric exchange and that the exchange occurs by a stepwise proton-transfer mechanism via a hydrogen bridge.
Assuntos
Porfirinas/química , Calorimetria , Isomerismo , Conformação Molecular , Estrutura Molecular , Porfirinas/síntese química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.
Assuntos
Proteínas da Matriz Extracelular/genética , Heparina/metabolismo , Trombospondinas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Escherichia coli , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Substâncias de Crescimento/química , Substâncias de Crescimento/genética , Substâncias de Crescimento/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Trombospondinas/metabolismoRESUMO
The epidural administration is used commonly in the treatment of pain. Nonsteroidal anti-inflammatory drugs, especially ibuprofen, would have potential in epidural use. Like many epidurally useful drugs it, however, has a short duration of action, which is a limiting factor. To improve epidural pain treatment, a long-acting, single-dose gel injection is being developed. In the present study, the possibility of using liposomal systems to control the release and dural permeation of ibuprofen was investigated in vitro. Liposomal solutions of ibuprofen.Na (20 mg/ml) were prepared by high-pressure homogenization from egg phosphatidylcholine. The liposomal gel consisted of poloxamer 407 and the liposomal solution. No signs in the 1H-NMR spectroscopy of line broadenings or chemical shifts were observed. The liposomal formulations were reproducible and stable. Ibuprofen release in phosphate buffer, pH 7.4, at 37 degrees C from the liposomal solution and the liposomal gel were prolonged significantly compared with their respective solution and gel controls. The liposomal gel controlled ibuprofen release and dural permeation in vitro and showed a permeation pattern favourable for maintaining constant drug levels. The liposomal poloxamer gel represents a new formulation approach to increase the local epidural availability of ibuprofen. It appeared to be a promising injectable controlled-release drug delivery system.
Assuntos
Anti-Inflamatórios não Esteroides/química , Ibuprofeno/química , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Portadores de Fármacos , Estabilidade de Medicamentos , Dura-Máter/metabolismo , Géis , Ibuprofeno/metabolismo , Técnicas In Vitro , Lipossomos , Espectroscopia de Ressonância Magnética , Permeabilidade , Fosfatidilcolinas , Poloxâmero , Solubilidade , SuínosRESUMO
Levosimendan, an inodilatory drug discovered using troponin C as a target protein, has a cardiac effect deriving from the calcium sensitization of contractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amino acid residues on helixepsilon of the N-terminal domain of this calcium-binding protein. Nine organic molecules, obtained by chemical modification of levosimendan, were tested both for their calcium-dependent binding to troponin C and troponin complex affinity HPLC columns, and for their ability to increase the calcium sensitivity of myofilaments in cardiac skinned fibers. A good correlation between the calcium sensitization and the calcium-dependent binding to troponin complex (r=0.90) and to cardiac troponin C (r=0.91) for the analogs of levosimendan was shown. In addition, the effect of levosimendan on the calcium-induced conformational changes in native and point-mutated cTnC was studied. Cys84-->Ser, Asp87-->Lys and Asp88-->Ala point-mutated cTnC were shown to maintain a high affinity to calcium, but their Ca(2+)titration curves were not influenced by levosimendan as for the native protein. Finally, it was demonstrated that the NMR chemical shifts of the terminal methyl groups of Met47, Met81, and Met85 on calcium-saturated cTnC were changed after addition of levosimendan in water solution at pH 7.4. This effect was not seen when adding an analog of levosimendan, which did not bind to the troponin C affinity HPLC column and did not increase the calcium-induced tension in cardiac skinned fibers.
Assuntos
Cálcio/metabolismo , Hidrazonas/metabolismo , Piridazinas/metabolismo , Troponina C/metabolismo , Vasodilatadores/metabolismo , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Hidrazonas/química , Estrutura Molecular , Miocárdio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Piridazinas/química , Simendana , Relação Estrutura-Atividade , Troponina C/genética , Vasodilatadores/químicaRESUMO
Two methods for the measurement of homonuclear 3J(HNH)alpha coupling constants are described. Both HSQC- and HMQC-type experiments employ 'quantitative J-correlation', in which the coupling constant of interest is obtained from the intensity ratio of cross peaks of two spectra. The first spectrum is acquired with 3J(HNH)alpha evolution and the second with alpha-proton decoupling. The resolution of these methods in the F1-domain is not restricted.