RESUMO
A 2-naphthol derivative 2 corresponding to the aromatic ring moiety of neocarzinostatin chromophore was found to degrade proteins under photo-irradiation with long-wavelength UV light without any additives under neutral conditions. Structure-activity relationship studies of the derivative revealed that methylation of the hydroxyl group at the C2 position of 2 significantly suppressed its photodegradation ability. Furthermore, a purpose-designed synthetic tumor-related biomarker, a H2 O2 -activatable photosensitizer 8 possessing a H2 O2 -responsive arylboronic ester moiety conjugated to the hydroxyl group at the C2 position of 2, showed significantly lower photodegradation ability compared to 2. However, release of the 2 from 8 by reaction with H2 O2 regenerated the photodegradation ability. Compound 8 exhibited selective photo-cytotoxicity against high H2 O2 -expressing cancer cells upon irradiation with long-wavelength UV light.
Assuntos
Naftóis , Proteínas , Zinostatina/análogos & derivados , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/química , Camundongos , Naftóis/química , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/toxicidade , Proteínas/efeitos dos fármacos , Proteínas/efeitos da radiação , Zinostatina/química , Zinostatina/toxicidadeRESUMO
Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzinostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the ß sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corroborate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic modalities towards targeted cancer therapy.
Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/ultraestrutura , Aptâmeros de Nucleotídeos/química , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Zinostatina/química , Sítios de Ligação , Citotoxinas , Molécula de Adesão da Célula Epitelial , Conformação Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas/métodosRESUMO
Neocarzinostatin (NCS) consists of an enediyne chromophore and an apoprotein (NCP). Lidamycin (LDM) is composed of another active enediyne chromophore (AE) and an acidic protein (LDP). Although the structures of NCP and LDP are very similar, LDM has been shown to have an increased tumor-suppressive activity than that of NCS. The aim of this study was to construct a chimeric protein (CMP) that consists of both the terminus residue of NCP and an LDP pocket-forming residue that can bind AE. This CMP will have a structure similar to NCS and an antitumor activity similar to LDM. The assembling efficiency of LDP, CMP, and NCP was 73.9, 1.5, and 1.1%, respectively. The cytotoxicity was consistent with their assembling efficiency of AE in proteins. When CMP-AE and NCP-AE were administered at equivalent AE doses of LDM, the inhibition rate of CMP-AE was the same as LDM and significantly higher than that of NCP-AE. Our study implied that the binding activity between LDP and AE was very specific. The terminus residue of LDP could affect the specifically binding activity. The pocket-forming residue could confer a protective function to the chromophore. Further investigation of its bioactivity might serve as a new drug design strategy and drug-delivery carrier in targeted cancer therapy.
Assuntos
Aminoglicosídeos/química , Antineoplásicos/química , Apoproteínas/química , Enedi-Inos/química , Proteínas Recombinantes de Fusão/química , Zinostatina/química , Aminoglicosídeos/genética , Animais , Antineoplásicos/farmacologia , Apoproteínas/genética , Apoproteínas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N'(17'-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called 'Trojan horse' strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (kcat/KM=13.6M(-1)s(-1) at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the 'Trojan horse' strategy for obtaining better catalysts of selective reactions will be further studied.
Assuntos
Biocatálise , Compostos Organometálicos/metabolismo , Ribonucleases/metabolismo , Zinco/metabolismo , Zinostatina/metabolismo , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/química , Zinco/química , Zinostatina/químicaRESUMO
Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role.
Assuntos
Antibióticos Antineoplásicos/química , Cafeína/química , Sondas de DNA/química , DNA/química , Zinostatina/química , Sequestradores de Radicais Livres/química , Radicais Livres/química , Substâncias Intercalantes/química , Cinética , Mutagênicos/química , Zinostatina/análogos & derivadosRESUMO
Neocarzinostatin is an antibiotic chromoprotein produced by Streptomyces carzinostaticus. Its enediyne-containing chromophore exhibits high DNA cleavage activity and belongs to one of the most potent categories of antitumor agents. The labile chromophore is readily inactivated by environmental thiols including the most abundant glutathione. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are of interest. Site-directed mutagenesis studies of the neocarzinostatin protein have shown that residues D33 and D99 play primary and secondary roles, respectively, in preserving neocarzinostatin from acidic glutathione whereas D79 and other residues around the opening of the binding cleft have an insignificant effect. Biothiol analyses revealed that cells of S. carzinostaticus produced no glutathione, but instead neutral mycothiol, which is known to serve functions analogous to glutathione. Mycothiol was the only neutral-charged thiol produced by the organism; all other identified biothiols carried at least partial negative charges. When the bacteria were cultured under conditions that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly, which suggests mycothiol-dependent cellular detoxification. Treating neocarzinostatin samples with the cell extract that retained active sulfhydryls led to efficient drug inactivation, which indicates that mycothiol is allowed to approach the protein-bound chromophore. The anionic side-chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol-screening operation: Preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge-charge repulsion in the outer-cell environment and detoxifying the toxin in the inner-cell body for self-resistance by accepting the cell-produced neutral mycothiol.
Assuntos
Antibacterianos/química , Enedi-Inos/química , Streptomyces/química , Compostos de Sulfidrila/análise , Zinostatina/química , Antibacterianos/metabolismo , Cisteína/metabolismo , Enedi-Inos/metabolismo , Glutationa/química , Glutationa/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Estrutura Molecular , Streptomyces/metabolismo , Compostos de Sulfidrila/química , Zinostatina/biossíntese , Zinostatina/metabolismoRESUMO
The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.
Assuntos
Proteínas de Transporte/química , Cisteína/química , Enedi-Inos/química , Zinostatina/química , Antibióticos Antineoplásicos/química , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Compostos de Sulfidrila/químicaRESUMO
The enediynes, unified by their unique molecular architecture and mode of action, represent some of the most potent anticancer drugs ever discovered. The biosynthesis of the enediyne core has been predicted to be initiated by a polyketide synthase (PKS) that is distinct from all known PKSs. Characterization of the enediyne PKS involved in C-1027 (SgcE) and neocarzinostatin (NcsE) biosynthesis has now revealed that (i) the PKSs contain a central acyl carrier protein domain and C-terminal phosphopantetheinyl transferase domain; (ii) the PKSs are functional in heterologous hosts, and coexpression with an enediyne thioesterase gene produces the first isolable compound, 1,3,5,7,9,11,13-pentadecaheptaene, in enediyne core biosynthesis; and (iii) the findings for SgcE and NcsE are likely shared among all nine-membered enediynes, thereby supporting a common mechanism to initiate enediyne biosynthesis.
Assuntos
Proteína de Transporte de Acila/química , Antibióticos Antineoplásicos/biossíntese , Enedi-Inos/metabolismo , Polienos/química , Policetídeo Sintases/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Aminoglicosídeos/biossíntese , Aminoglicosídeos/química , Sítios de Ligação , Biologia Computacional , Enedi-Inos/química , Dados de Sequência Molecular , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/química , Policetídeo Sintases/genética , Estrutura Terciária de Proteína , Zinostatina/biossíntese , Zinostatina/químicaRESUMO
The nine-membered enediyne class has drawn extensive interest because of extremely high antitumor potency and intricate interactions with its carrier protein. While the drug-induced DNA cleavage reactions have been mostly elucidated, the critical release-transport process of the labile enediyne molecule in cellular environment remained obscure. Using neocarzinostatin chromoprotein as a model, we demonstrated a lipid bilayer-assisted release mechanism. The in vitro enediyne release rate under aqueous conditions was found to be too slow to account for its efficient DNA cleavage action. Via the presence of lipid bilayers, chaotropic agents, or organic solvents, we found the release was substantially enhanced. The increased rate was linearly dependent on the lipid bilayer concentration and the dielectric value of the binary organic solvent mixtures. While lipid bilayers provided a low surrounding dielectricity to assist in drug release, there were no major conformational changes in the apo and holo forms of the carrier protein. In addition, the lifespan of the released enediyne chromophore was markedly extended through partitioning of the chromophore in the hydrophobic bilayer phase, and the lipid bilayer-stabilized enediyne chromophore significantly enhanced DNA cleavage in vitro. Collectively, we depicted how a lipid bilayer membrane efficiently enhanced dissociation of the enediyne chromophore through a hydrophobic sensing release mechanism and then acted as a protector of the released enediyne molecule until its delivery to the target DNA. The proposed membrane-assisted antibiotic release-transport model might signify a new dimension to our understanding of the modus operandi of the antitumor enediyne drugs.
Assuntos
Antibióticos Antineoplásicos/química , Enedi-Inos/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Zinostatina/química , Antibióticos Antineoplásicos/metabolismo , Clivagem do DNA , Enedi-Inos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Espectrometria de Fluorescência , Zinostatina/metabolismoRESUMO
The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein+chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.
Assuntos
Simulação de Dinâmica Molecular , Zinostatina/química , Zinostatina/metabolismo , Estrutura Secundária de Proteína , Zinostatina/análogos & derivadosRESUMO
Neocarzinostatin (NCS) is an antitumor chromophore carrier protein with many therapeutic applications. To characterize its binding and release mechanism, we have carried out molecular dynamics (MD) simulations for apo-NCS and holo-NCS. Although the beta-sheet regions of the protein exhibit restricted motion, large atomic fluctuations were observed in the loop regions, especially loop 99-104. This loop undergoes a "door-opening" motion that can facilitate chromophore binding and release. Calculated NMR order parameters confirm the simulated loop flexibility. We also provide a proposed explanation for the release rate difference for two mutants F78L and F78A through our simulation. The binding site structures of holo-NCS were also validated by chemical shift perturbations. Based on these results, a new binding and release mechanism for the NCS chromophore is proposed.
Assuntos
Antineoplásicos/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Zinostatina/análogos & derivados , Zinostatina/químicaRESUMO
Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications.The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques.
Assuntos
Antineoplásicos/química , Dissulfetos/química , Zinostatina/química , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Sequência de Bases , Simulação por Computador , Cristalografia por Raios X , Evolução Molecular Direcionada , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Testosterona/química , Termodinâmica , Zinostatina/metabolismoRESUMO
BACKGROUND: Neocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier. METHODS: We characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy. RESULTS: The partially unfolded protein is in molten globule-like state, in which approximately 60% and approximately 20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state. CONCLUSION: These findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier.
Assuntos
Antineoplásicos/administração & dosagem , Apoproteínas/química , Portadores de Fármacos/química , Enedi-Inos/administração & dosagem , Zinostatina/química , Antineoplásicos/química , Sítios de Ligação , Dicroísmo Circular , Enedi-Inos/química , Guanidina/farmacologia , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Structures of the protein-chromophore complex and the apoprotein form of neocarzinostatin were determined at 1.8 angstrom resolution. Neocarzinostatin is composed of a labile chromophore with DNA-cleaving activity and a stabilizing protein. The chromophore displays marked nonlinearity of the triple bonds and is bound noncovalently in a pocket formed by the two protein domains. The chromophore pi-face interacts with the phenyl ring edges of Phe52 and Phe78. The amino sugar and carbonate groups of the chromophore are solvent exposed, whereas the epoxide, acetylene groups, and carbon C-12, the site of nucleophilic thiol addition during chromophore activation, are unexposed. The position of the amino group of the chromophore carbohydrate relative to C-12 supports the idea that the amino group plays a role in thiol activation.
Assuntos
Zinostatina/química , Apoproteínas/química , Gráficos por Computador , Simulação por Computador , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Neocarzinostatin chromophore (NCS chrom) was found to induce site-specific cleavage at the 3' side of a bulge in single-stranded DNA in the absence of thiol. This reaction involved the oxidative formation of a DNA fragment with a nucleoside 5'-aldehyde at its 5' terminus and generated an ultraviolet light-absorbing and fluorescent species of post-activated drug containing tritium abstracted from the carbon at the 5' position of the target nucleotide. The DNAs containing point mutations that disrupt the bulge were not cleavage substrates and did not generate this drug product. Thus, DNA is an active participant in its own destruction, and NCS chrom may be useful as a probe for bulged structures in nucleic acids.
Assuntos
Dano ao DNA , DNA de Cadeia Simples/efeitos dos fármacos , Zinostatina/análogos & derivados , Sequência de Bases , Biotransformação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Enedi-Inos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oxirredução , Piperidinas/farmacologia , Mutação Puntual , Compostos de Sulfidrila/farmacologia , Zinostatina/química , Zinostatina/metabolismo , Zinostatina/farmacologiaRESUMO
Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.
Assuntos
DNA/metabolismo , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Composição de Bases , Sequência de Bases , DNA/química , Enedi-Inos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Zinostatina/análogos & derivados , Zinostatina/química , Zinostatina/metabolismoRESUMO
Bulged sites in DNA and RNA have become targets for rational drug design due to their suspected involvement in a number of key biomolecular processes. A lead compound, derived from the enediyne natural product NCS-chrom has been used to inform chemical synthesis of a family of designed probes of DNA bulges, one of which shows 80 nM affinity for a two base bulged target. Key contributors to binding of these spirocyclic compounds have been studied in order to correlate affinity and specificity with structural features. Herein, we demonstrate that the glycosyl linkage stereochemistry of the pendant aminofucosyl group plays a pivotal role in binding, and coupled with insight obtained with various bulged targets, will allow rational design of second generation ligands.
Assuntos
Sondas de DNA , Zinostatina/química , Sequência de Bases , Fluorescência , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the protein-bound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Zinostatina/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Etanol/farmacologia , Etídio/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína , Reprodutibilidade dos Testes , Termodinâmica , Temperatura de Transição/efeitos dos fármacos , Ureia/farmacologia , Zinostatina/químicaRESUMO
We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.
Assuntos
Evolução Molecular , Testosterona/metabolismo , Zinostatina/química , Zinostatina/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Técnicas In Vitro , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
[reaction: see text] A key structural feature of the Neocarzinostatin chromophore is a reactive epoxydiyne. We present here a new method for the preparation of epoxydiynes by the addition of an allenyl zinc bromide to a propargylic ketone.