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1.
Anal Chem ; 91(23): 14936-14942, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31670502

RESUMO

Förster resonance energy transfer (FRET) is a well-established method for studying macromolecular interactions and conformational changes within proteins. Such a method normally uses fluorescent proteins or chemical-labeling methods which are often only accessible to surface-exposed residues and risk-disturbing target protein structures. Here, we demonstrate that the genetic incorporation of a synthetic fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) and natural endogenous fluorophore Tryptophan (Trp) residues of a protein could serve as an efficient FRET pair to monitor protein interactions, using the signaling transducer ß-arrestin-1 as a model system. We used this technology to record the dynamic spectra in both binding and competition experiments of ß-arrestin-1, the contribution of each specific phosphate in ternary complex formation, in a rapid and efficient manner. The determined Kd value for the association between the active arrestin and Fab30 is 0.68 µM in the three-component interaction system. Moreover, we were able to determine the contributions of the site 3 phospho-site and the site 6 phospho-site binding, each contributing to the high affinity ternary complex assembly as 2.7 fold and 15.5 fold, respectively, which were never determined before. These results thus highlighted the potential usage of this new method in measurement of the allosteric-induced enhanced affinity with small amount proteins and in a fast manner and in a complex system. Collectively, our newly developed Trp:Cou FRET system based on genetic expansion technology has extended the molecular toolboxes available for biochemical and structural biology studies.

2.
J Neurochem ; 148(4): 550-560, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30451284

RESUMO

Protein Phosphatase Mg2+ /Mn2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.


Assuntos
Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/genética , Biocatálise , Humanos , Mutação , Relação Estrutura-Atividade
3.
Nat Chem Biol ; 14(9): 876-886, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30120361

RESUMO

Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote ß-arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor-arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conformational states of ß-arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling.


Assuntos
Regulação Alostérica , Arrestina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Domínios de Homologia de src , Células Cultivadas , Células HEK293 , Humanos
4.
Zhongguo Zhong Xi Yi Jie He Za Zhi ; 31(6): 745-8, 2011 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-21823415

RESUMO

OBJECTIVE: To study the effect on the motor function of stroke patients by combination of needling at Back-shu point and trunk exercise. METHODS: Ninety stroke hemiplegic patients were randomly assigned to the conventional treatment group (as the convention group), the Back-shu point needling group, and the combination of Back-shu point needling and the trunk exercise group, 30 patients in each group. They were treated with the conventional treatment, needling at Back-shu point, and the combination of needling at Back-shu point and trunk exercise. The Fugl-Meyer score (FMA) and modified Barthel index (MBI) score were assessed before treatment and two months after treatment. RESULTS: The three rehabilitation treatment methods were all effective in improving the motor function of stroke hemiplegic patients (P<0.05). The effects in the Back-shu point needling group and the combination of Back-shu point needling and the trunk exercise group were respectively superior to that in the conventional treatment group (P<0.05). The effect in the combination of Back-shu point needling and the trunk exercise group was superior to that in the Back-shu point needling group (P<0.05). CONCLUSION: The combination of Back-shu point needling and the trunk exercise could improve the motor function of stroke hemiplegic patients, and its effect was better than needling at Back-shu point alone.


Assuntos
Terapia por Acupuntura , Reabilitação do Acidente Vascular Cerebral , Acidente Vascular Cerebral/terapia , Idoso , Terapia por Exercício , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/fisiopatologia
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