Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Angew Chem Int Ed Engl ; : e202408979, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38979660

RESUMO

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising-strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via the widely used Strep-tag II affinity peptide. Electrochemistry, surface-sensitive spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tag labeled enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology.

2.
Sci Rep ; 13(1): 16387, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37773489

RESUMO

New variants of SARS-CoV-2 that can escape immune response continue to emerge. Consequently, there is an urgent demand to design small molecule therapeutics inhibiting viral entry to host cells to reduce infectivity rate. Despite numerous in silico and in situ studies, the structural requirement of designing viral-entry inhibitors effective against multiple variants of SARS-CoV-2 has yet to be described. Here we systematically screened the binding of various natural products (NPs) to six different SARS-CoV-2 receptor-binding domain (RBD) structures. We demonstrate that Multi-structural Molecular Docking (MOD) combined with molecular dynamics calculations allowed us to predict a vulnerable site of RBD and the structural requirement of ligands binding to this vulnerable site. We expect that our findings lay the foundation for in silico screening and identification of lead molecules to guide drug discovery into designing new broad-spectrum lead molecules to counter the threat of future variants of SARS-CoV-2.


Assuntos
Produtos Biológicos , COVID-19 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2 , Ligação Proteica
3.
Nat Commun ; 13(1): 4843, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35977922

RESUMO

Protein halogenation is a common non-enzymatic post-translational modification contributing to aging, oxidative stress-related diseases and cancer. Here, we report a genetically encodable halogenation of tyrosine residues in a reconstituted prokaryotic filamentous cell-division protein (FtsZ) as a platform to elucidate the implications of halogenation that can be extrapolated to living systems of much higher complexity. We show how single halogenations can fine-tune protein structures and dynamics of FtsZ with subtle perturbations collectively amplified by the process of FtsZ self-organization. Based on experiments and theories, we have gained valuable insights into the mechanism of halogen influence. The bending of FtsZ structures occurs by affecting surface charges and internal domain distances and is reflected in the decline of GTPase activities by reducing GTP binding energy during polymerization. Our results point to a better understanding of the physiological and pathological effects of protein halogenation and may contribute to the development of potential diagnostic tools.


Assuntos
Proteínas de Bactérias , Proteínas do Citoesqueleto , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Halogenação , Ligação Proteica , Tirosina/metabolismo
5.
Front Microbiol ; 13: 1073315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36733774

RESUMO

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel-iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes.

6.
J Phys Chem B ; 125(34): 9668-9677, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34427096

RESUMO

Cytochrome c oxidase (CcO) pumps protons from the N-side to the P-side and consumes electrons from the P-side of the mitochondrial membrane driven by energy gained from reduction of dioxygen to water. ATP synthesis uses the resulting proton gradient and electrostatic potential difference. Since the distance a proton travels through CcO is too large for a one-step transfer process, proton-loading sites (PLS) that can carry protons transiently are necessary. One specific pump-active PLS couples to the redox reaction, thus energizing the proton to move across the membrane against electric potential and proton gradient. The PLS should also prevent proton backflow. Therefore, the propionates of the two redox-active hemes in CcO were suggested as PLS candidates although, according to CcO crystal structures, none of the four propionates can be protonated on account of strong H-bonds. Here, we show that modeling the local structure around heme a3 propionates enhances significantly their capability of carrying a proton jointly. This was not possible for the propionates of heme a. The modeled structures are stable in molecular dynamics simulations (MDS) and are energetically similar to the crystal structure. Precise electrostatic energy computations of MDS data are used to estimate the pKA values of all titratable residues in CcO. For the modeled structures, the heme a3 propionates have pKA values high enough to host a proton transiently but not too high to fix the proton permanently. The change in pKA throughout the redox reaction is sufficient to push the proton to the P-side of the membrane and to provide the protons with the necessary amount of energy for ATP synthesis.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Prótons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Heme/metabolismo , Oxirredução , Propionatos , Bombas de Próton/metabolismo
7.
Chembiochem ; 22(20): 2946-2950, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34265150

RESUMO

Since the emergence of SARS-CoV-2, little attention has been paid to the interplay between the interaction of virus and commensal microbiota. Here, we used molecular docking and dynamics simulations to study the interaction of some of the known metabolites and natural products (NPs) produced by commensal microbiota with the receptor binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. The results predict that NPs of commensal microbiota such as bile acids and non-ribosomal peptides (NRPs), of which some are siderophores, bind to the wild-type RBD and interfere with its binding to the ACE2 receptor. N501Y mutation, which is present in many of the emerging variants of the virus, abolishes the predicted binding pocket of bile acids and NRPs. Based on these findings, available experimental data showing that bile acids reduce the binding affinity of wild-type RBD to the ACE2 receptor, and the data suggesting that the respiratory tract microbiota affect viral infection we put forward the following proposal: mutations such as N501Y enable the RBD to bind to the ACE2 receptor more effectively in the presence of NPs produced by the respiratory tract bacteria thereby, increasing the infectivity rate of the virus. We hope our data stimulate future works to better understand the interactions of NPs produced by commensal microbiota with respiratory viruses like SARS-CoV-2.


Assuntos
Produtos Biológicos , COVID-19/genética , COVID-19/virologia , Variação Genética/genética , Microbiota , SARS-CoV-2/genética , Animais , Bactérias/metabolismo , Produtos Biológicos/metabolismo , Simulação por Computador , Humanos , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/metabolismo
8.
Front Chem ; 9: 669452, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33987170

RESUMO

Cytochrome c oxidase (CcO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O2 reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CcO from Rhodobacter sphaeroides. The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (R4CO, R2CO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from R4CO to R2CO. We performed potential jump experiments to accumulate the R2CO and R4CO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a3. Our experiments also suggest propionate A of heme a3 changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site.

9.
J Am Chem Soc ; 143(7): 2769-2776, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33560128

RESUMO

Cytochrome c oxidase (CcO) in its as-isolated form is known to exist in a slow and fast form, which differ drastically in their ability to bind oxygen and other ligands. While preparation methods have been established that yield either the fast or the slow form of the protein, the underlying structural differences have not been identified yet. Here, we have performed surface enhanced resonance Raman (SERR) spectroscopy of CcO immobilized on electrodes in both forms. SERR spectra obtained in resonance with the heme a3 metal-to-ligand charge transfer (MLCT) transition at 650 nm displayed a sharp vibrational band at 748 or 750 cm-1 when the protein was in its slow or fast form, respectively. DFT calculations identified the band as a mode of the His-419 ligand that is sensitive to the oxygen ligand and the protonation state of Tyr-288 within the binuclear complex. Potential-dependent SERR spectroscopy showed a redox-induced change of this band around 525 mV versus Ag/AgCl exclusively for the fast form, which coincides with the redox potential of the Tyr-O/Tyr-O- transition. Our data points to a peroxide ligand in the resting state of CcO for both forms. The observed frequencies and redox sensitivities of the Raman marker band suggest that a radical Tyr-288 is present in the fast form and a protonated Tyr-288 in the slow form.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Imobilizadas , Análise Espectral Raman , Teoria da Densidade Funcional , Técnicas Eletroquímicas , Eletrodos , Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Ligantes , Oxigênio/química , Oxigênio/metabolismo , Rhodobacter sphaeroides/metabolismo
10.
Sci Rep ; 10(1): 6853, 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32300182

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

11.
Sci Rep ; 10(1): 3863, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123230

RESUMO

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a3 to heme a.


Assuntos
Proteínas de Bactérias/química , Cianatos/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Rhodobacter sphaeroides/enzimologia , Domínio Catalítico
12.
Proteins ; 86(4): 439-453, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29344998

RESUMO

Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal-dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg2+ -ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction-competent conformation. Active-site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1-DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Sítios de Ligação , Domínio Catalítico , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Prótons , Especificidade por Substrato
13.
Phys Chem Chem Phys ; 19(47): 32143, 2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29159336

RESUMO

Retraction of 'The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation' by Hendrik Mohrmann et al., Phys. Chem. Chem. Phys., 2017, DOI: .

14.
Phys Chem Chem Phys ; 2017 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-29067359

RESUMO

Cytochrome c oxidase (CcO) is a membrane protein of the respiratory chain that catalytically reduces molecular oxygen (O2) to water while translocating protons across the membrane. The enzyme hosts two copper and two heme iron moieties (heme a/heme a3). The atomic details of the sequential steps that go along with this redox-driven proton translocation are a matter of debate. Particularly for the reductive phase of CcO that precedes oxygen binding experimental data are scarce. Here, we use CcO under anaerobic conditions where carbon monoxide (CO) is bound to heme a3 which in tandem with CuB forms the binuclear center (BNC). Fourier-transform infrared (FTIR) absorption spectroscopy is combined with electro-chemistry to probe different redox and protonation states populated by variation of the external electrostatic potential. With this approach, the redox behavior of heme a and the BNC could be separated and the corresponding redox potentials were determined. We also infer the protonation of one of the propionate side chains of heme a3 to correlate with the oxidation of heme a. Experimental changes in the local electric field surrounding CO bound to heme a3 are determined by their vibrational Stark effect and agree well with electrostatic computations. The comparison of experimental and computational results indicates that changes of the heme a3/CuB redox state are coupled to proton transfer towards heme a3. The latter supports the role of the heme a3 propionate D as proton loading site.

15.
Photochem Photobiol ; 93(3): 713-723, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28500721

RESUMO

Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin-chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process. Although the three-dimensional structure of Agp1 has been solved at high resolution, the precise position of hydrogen atoms and protonation pattern in the chromophore binding pocket has not been investigated yet. Here, we present protonated structure models of Agp1 in the Pr state involving appropriately placed hydrogen atoms that were generated by hybrid quantum mechanics/molecular mechanics- and electrostatic calculations and validated against experimental structural- and spectroscopic data. Although the effect of histidine protonation on the vibrational spectra is weak, our results favor charge neutral H250 and H280 both protonated at Nε. However, a neutral H250 with a proton at Nε and a cationic H280 may also be possible. Furthermore, the present QM/MM calculations of IR and Raman spectra of Agp1 containing isotope-labeled BV provide a detailed vibrational assignment of the biliverdin modes in the fingerprint region.


Assuntos
Proteínas de Bactérias/química , Fitocromo/química , Conformação Proteica , Vibração , Sítios de Ligação , Modelos Químicos , Eletricidade Estática
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...