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1.
Sci Adv ; 10(24): eadn8386, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38865454

RESUMO

Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15-Z,anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15-E,syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.


Assuntos
Luz , Cianobactérias/metabolismo , Cianobactérias/fisiologia , Aclimatação , Fotossíntese , Fitocromo/metabolismo , Fitocromo/química , Modelos Moleculares , Pigmentos Biliares/metabolismo , Pigmentos Biliares/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Luz Vermelha
2.
Biochem Biophys Res Commun ; 625: 87-93, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35952612

RESUMO

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.


Assuntos
Drosophila , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Drosophila/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Núcleo Familiar , Peptídeos/metabolismo , Prolina/metabolismo , Ligação Proteica , Proteínas Son Of Sevenless/metabolismo
3.
Int J Mol Sci ; 23(4)2022 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-35216169

RESUMO

Saponaria officinalis L., commonly known as "Soapwort", is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1-17), including seven new glycosides (1-7). The structures of 1-7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.


Assuntos
Antineoplásicos/toxicidade , Apoptose , Mitocôndrias/metabolismo , Ácido Oleanólico/toxicidade , Saponaria/química , Células A549 , Ciclo Celular/efeitos dos fármacos , Humanos , Ácido Oleanólico/metabolismo , Saponaria/metabolismo , Sementes/química , Sementes/metabolismo
4.
J Phys Chem B ; 126(4): 813-821, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35076228

RESUMO

Cyanobacteriochromes (CBCRs) belong to the phytochrome superfamily of photoreceptors, the members of which utilize a linear tetrapyrrole (bilin) as a chromophore. RcaE is a representative member of a green/red-type CBCR subfamily that photoconverts between a green-absorbing dark state and red-absorbing photoproduct (Pr). Our recent crystallographic study showed that the phycocyanobilin (PCB) chromophore of RcaE adopts a unique C15-E,syn configuration in the Pr state, unlike the typical C15-E,anti configuration for the phytochromes and other CBCRs. Here, we measured Raman spectra of the Pr state of RcaE with 1064 nm excitation and explored the structure of PCB and its interacting residues under physiologically relevant aqueous conditions. We also performed measurements of RcaE in D2O as well as the sample reconstituted with the PCB labeled with 15N or with both 13C and 15N. The observed Raman spectra were analyzed by quantum mechanics/molecular mechanics (QM/MM) calculations together with molecular dynamics simulations. The Raman spectra and their isotope effects were well-reproduced by the simulated spectra of fully protonated PCB with the C15-E,syn configuration and allowed us to assign most of the observed bands. The present vibrational analysis of the all syn bilin chromophore using the QM/MM method will advance future studies on CBCRs and the related proteins by vibrational spectroscopy.


Assuntos
Fotorreceptores Microbianos , Fitocromo , Proteínas de Bactérias/química , Pigmentos Biliares/química , Simulação de Dinâmica Molecular , Fotorreceptores Microbianos/química , Fitocromo/química , Análise Espectral Raman
5.
Dalton Trans ; 50(36): 12716-12722, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34545880

RESUMO

A series of novel metal complexes were synthesized containing an Ir-cyclometalated bichromophore as a visible-light sensitizer. A new bichromophoric unit containing a naphthyl substituent and methyl substituents on the 2-phenylpyridine chelating ligand was synthesized and characterized for the first time. According to the increased crystallinity of the bichromophoric unit, novel Ir-M metal complexes (M = Pd, Mn, and Ir) were synthesized and fully characterized. The novel Ir-Pd complex maintained photocatalytic activity toward styrenes under visible-light irradiation, and polymerization with p-chlorostyrene, copolymerization with styrene and p-chlorostyrene furnished corresponding polymers.

7.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33972439

RESUMO

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the ß-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.


Assuntos
Proteínas de Bactérias/química , Pigmentos Biliares/química , Complexos de Proteínas Captadores de Luz/química , Fotorreceptores Microbianos/química , Fitocromo/química , Prótons , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pigmentos Biliares/genética , Pigmentos Biliares/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cianobactérias/química , Cianobactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Luz , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Simulação de Dinâmica Molecular , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pirróis/química , Pirróis/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Nat Commun ; 11(1): 4916, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004803

RESUMO

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.


Assuntos
Brassica rapa/fisiologia , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Animais , Cristalografia , Flores/metabolismo , Haplótipos , Simulação de Dinâmica Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Pólen/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera
9.
Biochim Biophys Acta Gen Subj ; 1864(2): 129419, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31449838

RESUMO

Structural study of multidomain proteins using NMR is an emerging issue for understanding biological functions. To this end, domain-specific labeling is expected to be a key technology for facilitating the NMR-assignment process and for collecting distance information via spin labeling. To obtain domain-specific labeled samples, use of sortase A as a protein ligation tool is a viable approach. Sortase A enables ligation of separately expressed proteins (domains) through the Leu-Pro-X-Thr-Gly linker. However, the ligation reaction mediated by sortase A is not efficient. Poor yield and long reaction times hamper large-scale preparation using sortase A. Here we report the application of highly active sortases to NMR analyses. Optimal yields can be achieved within several hours when the ligation reaction are mediated by highly active sortases at 4 °C. We propose that this protocol can contribute to structural analyses of multidomain proteins by NMR.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Staphylococcus aureus/enzimologia , Escherichia coli , Hidrólise , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Temperatura , Vinculina/química
10.
Angew Chem Int Ed Engl ; 58(22): 7284-7288, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-30938016

RESUMO

Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions, it is indispensable to observe their three-dimensional (3D) structures at the atomic level in a physiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/baculovirus system using NMR data from living cells exclusively. The method was applied to five proteins, rat calmodulin, human HRas, human ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein G B1 domain. In all cases, we could obtain structural information from well-resolved in-cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be a standard tool for protein structure determinations in living eukaryotic cells. For three proteins, we achieved well-converged 3D structures. Among these, the in-cell structure of protein G B1 domain was most accurately determined, demonstrating that a helix-loop region is tilted away from a ß-sheet compared to the conformation in diluted solution.


Assuntos
Algoritmos , Proteínas de Bactérias/química , Calmodulina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Proto-Oncogênicas p21(ras)/química , Ubiquitina/química , Animais , Humanos , Modelos Moleculares , Conformação Proteica em Folha beta , Ratos , Streptococcus/metabolismo , Thermus thermophilus/metabolismo
11.
Sci Rep ; 8(1): 10382, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29991771

RESUMO

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.


Assuntos
Proteínas Quinases/metabolismo , Ubiquitina/metabolismo , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , Humanos , Mutação , Transtornos Parkinsonianos/etiologia , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Quinases/química , Ubiquitina-Proteína Ligases/metabolismo
12.
Sci Rep ; 7(1): 12816, 2017 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-28993701

RESUMO

DJ-1 (also known as PARK7) has been identified as a causal gene for hereditary recessive Parkinson's disease (PD). Consequently, the full elucidation of DJ-1 function will help decipher the molecular mechanisms underlying PD pathogenesis. However, because various, and sometimes inconsistent, roles for DJ-1 have been reported, the molecular function of DJ-1 remains controversial. Recently, a number of papers have suggested that DJ-1 family proteins are involved in aldehyde detoxification. We found that DJ-1 indeed converts methylglyoxal (pyruvaldehyde)-adducted glutathione (GSH) to intact GSH and lactate. Based on evidence that DJ-1 functions in mitochondrial homeostasis, we focused on the possibility that DJ-1 protects co-enzyme A (CoA) and its precursor in the CoA synthetic pathway from aldehyde attack. Here, we show that intact CoA and ß-alanine, an intermediate in CoA synthesis, are recovered from methylglyoxal-adducts by recombinant DJ-1 purified from E. coli. In this process, methylglyoxal is converted to L-lactate rather than the D-lactate produced by a conventional glyoxalase. PD-related pathogenic mutations of DJ-1 (L10P, M26I, A104T, D149A, and L166P) impair or abolish detoxification activity, suggesting a pathological significance. We infer that a key to understanding the biological function of DJ-1 resides in its methylglyoxal-adduct hydrolase activity, which protects low-molecular thiols, including CoA, from aldehydes.


Assuntos
Aldeídos/metabolismo , Doença de Parkinson/metabolismo , Proteína Desglicase DJ-1/metabolismo , Compostos de Sulfidrila/metabolismo , Acetilcisteína/farmacologia , Sequência de Aminoácidos , Coenzima A/metabolismo , Glutationa/metabolismo , Células HeLa , Humanos , Inativação Metabólica/efeitos dos fármacos , Ácido Láctico/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Proteína Desglicase DJ-1/química , Proteína Desglicase DJ-1/genética , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , beta-Alanina/metabolismo
13.
Biomol NMR Assign ; 11(2): 123-126, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28284018

RESUMO

Negative regulator differentiation 1 (Nrd1), a fission yeast RNA binding protein, modulates cytokinesis and sexual development and contributes to stress granule formation in response to environmental stresses. Nrd1 comprises four RRM domains and binds and stabilizes Cdc4 mRNA that encodes the myosin II light chain. Nrd1 binds the Cpc2 fission-yeast RACK1 homolog, and the interaction promotes Nrd1 localization to stress granules. Interestingly, Pmk1 mitogen-activated protein kinase phosphorylates Thr40 in the unstructured N-terminal region and Thr126 in the first RRM domain of Nrd1. Phosphorylation significantly reduces RNA-binding activity and likely modulates Nrd1 function. To reveal the relationship between the structure and function of Nrd1 and how phosphorylation affects structure, we used heteronuclear NMR techniques to investigate the three-dimensional structure of Nrd1. Here we report the 1H, 13C, and 15N resonance assignments of RRM1-RRM2 (residues 108-284) comprising the first and second RRMs obtained using heteronuclear NMR techniques. Secondary structures derived from the chemical shifts are reported. These data should contribute to the understanding of the three-dimensional structure of the RRM1-RRM2 region of Nrd1 and the perturbation caused by phosphorylation.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Ribonucleoproteínas/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces , Estrutura Secundária de Proteína , Ribonucleoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
14.
Sci Rep ; 6: 38312, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27910948

RESUMO

Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 µM concentration in living E. coli cells.


Assuntos
Proteínas de Bactérias/química , Ressonância Magnética Nuclear Biomolecular/métodos , Receptores de IgG/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Teorema de Bayes , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus/genética , Streptococcus/metabolismo , Termodinâmica , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
15.
J Biomol NMR ; 66(2): 99-110, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27631409

RESUMO

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.


Assuntos
Quelantes/química , Elementos da Série dos Lantanídeos/química , Espectroscopia de Ressonância Magnética , Proteínas/química , Células HeLa , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Conformação Proteica , Estabilidade Proteica , Proteínas/genética , Ubiquitina/química , Ubiquitina/genética
17.
Biochem Biophys Res Commun ; 457(2): 200-5, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25545060

RESUMO

Despite their advantages in analysis, 4D NMR experiments are still infrequently used as a routine tool in protein NMR projects due to the long duration of the measurement and limited digital resolution. Recently, new acquisition techniques for speeding up multidimensional NMR experiments, such as nonlinear sampling, in combination with non-Fourier transform data processing methods have been proposed to be beneficial for 4D NMR experiments. Maximum entropy (MaxEnt) methods have been utilised for reconstructing nonlinearly sampled multi-dimensional NMR data. However, the artefacts arising from MaxEnt processing, particularly, in NOESY spectra have not yet been clearly assessed in comparison with other methods, such as quantitative maximum entropy, multidimensional decomposition, and compressed sensing. We compared MaxEnt with other methods in reconstructing 3D NOESY data acquired with variously reduced sparse sampling schedules and found that MaxEnt is robust, quick and competitive with other methods. Next, nonlinear sampling and MaxEnt processing were applied to 4D NOESY experiments, and the effect of the artefacts of MaxEnt was evaluated by calculating 3D structures from the NOE-derived distance restraints. Our results demonstrated that sufficiently converged and accurate structures (RMSD of 0.91Å to the mean and 1.36Å to the reference structures) were obtained even with NOESY spectra reconstructed from 1.6% randomly selected sampling points for indirect dimensions. This suggests that 3D MaxEnt processing in combination with nonlinear sampling schedules is still a useful and advantageous option for rapid acquisition of high-resolution 4D NOESY spectra of proteins.


Assuntos
Entropia , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Modelos Moleculares , Dinâmica não Linear , Reprodutibilidade dos Testes , Thermus thermophilus/metabolismo
18.
Sci Rep ; 4: 6016, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25125290

RESUMO

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.


Assuntos
Bombyx/genética , Bombyx/metabolismo , Larva/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Humanos , Larva/genética , Fosforilação , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/genética , Transdução de Sinais
19.
Structure ; 22(1): 35-46, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24268649

RESUMO

The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.


Assuntos
Caseína Quinase II/química , Proteínas de Homeodomínio/química , Proteínas Nucleares/química , Correpressor 2 de Receptor Nuclear/química , Sítios de Ligação , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Ligação a DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Fosforilação , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transcrição Gênica
20.
Biochem Biophys Res Commun ; 438(4): 653-9, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23933251

RESUMO

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.


Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteína G de Ligação ao Cálcio S100/metabolismo , Estresse Fisiológico , Calbindinas , Células HeLa , Humanos , Magnésio/metabolismo , Modelos Moleculares , Proteína G de Ligação ao Cálcio S100/análise
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