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1.
J Phys Chem Lett ; 11(5): 1940-1946, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32073271

RESUMO

We present vibrational and electronic photodissociation spectra of a model chromophore of the green fluorescent protein in complexes with up to two water molecules, prepared in a cryogenic ion trap at 160-180 K. We find the band origin of the singly hydrated chromophore at 20 985 cm-1 (476.5 nm) and observe partially resolved vibrational signatures. While a single water molecule induces only a small shift of the S1 electronic band of the chromophore, without significant change of the Franck-Condon envelope, the spectrum of the dihydrate shows significant broadening and a greater blue shift of the band edge. Comparison of the vibrational spectra with predicted infrared spectra from density functional theory indicates that water molecules can interact with the oxygen atom on the phenolate group or on the imidazole moiety, respectively.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Teoria da Densidade Funcional , Proteínas de Fluorescência Verde/química , Imidazóis/química , Oxigênio/química , Fenol/química , Água/química , Água/metabolismo
2.
Phys Chem Chem Phys ; 22(9): 4875-4879, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32072999

RESUMO

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E. coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Alquinos/química , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Óxidos de Nitrogênio/química , Marcadores de Spin
3.
Phys Chem Chem Phys ; 22(4): 2424-2428, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31939956

RESUMO

A strong π-donating group like p-NMe2 dramatically changes the excited-state behavior of the green fluorescent protein (GFP) chromophore, such as realizing charge-transfer absorption and executing significant photoinduced intramolecular charge transfer (ICT), which makes a planar first singlet (S1) excited-state minimum disappear and significantly lowers the S1 excited-state potential energy surface (PES), leading to barrierless τ-torsion and φ-torsion excited-state relaxation and eliciting the φ-torsion S1 excited-state minimum. This finding is critical since a strong π-donating group like p-NMe2 may do the same things to other fluorophores.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/efeitos da radiação , Luz , Eletricidade Estática
4.
J Chem Theory Comput ; 16(1): 587-600, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31815476

RESUMO

Quantum chemical calculations are important for elucidating light-capturing mechanisms in photobiological systems. The time-dependent density functional theory (TDDFT) has become a popular methodology because of its balance between accuracy and computational scaling, despite its problems in describing, for example, charge transfer states. As a step toward systematically understanding the performance of TDDFT calculations on biomolecular systems, we study here 17 commonly used density functionals, including seven long-range separated functionals, and compare the obtained results with excitation energies calculated at the approximate second order coupled-cluster theory level (CC2). The benchmarking set includes the first five singlet excited states of 11 chemical analogues of biochromophores from the green fluorescent protein, rhodopsin/bacteriorhodopsin (Rh/bR), and the photoactive yellow protein. We find that commonly used pure density functionals such as BP86, PBE, M11-L, and hybrid functionals with 20-25% of Hartree-Fock (HF) exchange (B3LYP, PBE0) have a tendency to consistently underestimate vertical excitation energies (VEEs) relative to the CC2 values, whereas hybrid density functionals with around 50% HF exchange such as BHLYP, PBE50, and M06-2X and long-range corrected functionals such as CAM-B3LYP, ωPBE, ωPBEh, ωB97X, ωB97XD, BNL, and M11 overestimate the VEEs. We observe that calculations using the CAM-B3LYP and ωPBEh functionals with 65% and 100% long-range HF exchange, respectively, lead to an overestimation of the VEEs by 0.2-0.3 eV for the benchmarking set. To reduce the systematic error, we introduce here two new empirical functionals, CAMh-B3LYP and ωhPBE0, for which we adjusted the long-range HF exchange to 50%. The introduced parameterization reduces the mean signed average (MSA) deviation to 0.07 eV and the root mean square (rms) deviation to 0.17 eV as compared to the CC2 values. In the present study, TDDFT calculations using the aug-def2-TZVP basis sets, the best performing functionals relative to CC2 are ωhPBE0 (rms = 0.17, MSA = 0.06 eV); CAMh-B3LYP (rms = 0.16, MSA = 0.07 eV); and PBE0 (rms = 0.23, MSA = -0.14 eV). For the popular range-separated CAM-B3LYP functional, we obtain an rms value of 0.31 eV and an MSA value of 0.25 eV, which can be compared with the rms and MSA values of 0.37 and -0.31 eV, respectively, as obtained at the B3LYP level.


Assuntos
Teoria da Densidade Funcional , Proteínas de Fluorescência Verde/química , Proteínas de Ligação ao Retinol/química , Algoritmos , Bases de Dados de Proteínas , Humanos , Modelos Moleculares
5.
J Phys Chem Lett ; 11(2): 492-496, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31880458

RESUMO

Neutron crystallography has been used to elucidate the protonation states for the enhanced green fluorescent protein, which has revolutionized imaging technologies. The structure has a deprotonated hydroxyl group in the fluorescent chromophore. Also, the protonation states of His148 and Thr203, as well as the orientation of a critical water molecule in direct contact with the chromophore, could be determined. The results demonstrate that the deprotonated hydroxyl group in the chromophore and the nitrogen atom ND1 in His148 are charged negatively and positively, respectively, forming an ion pair. The position of the two deuterium atoms in the critical water molecule appears to be displaced slightly toward the acceptor oxygen atoms according to their omit maps. This displacement implies the formation of an intriguing electrostatic potential realized inside of the protein. Our findings provide new insights into future protein design strategies along with developments in quantum chemical calculations.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas Mutantes/química , Prótons , Cristalografia por Raios X , Modelos Moleculares , Proteínas Mutantes/genética , Mutação , Eletricidade Estática
6.
Biosci Biotechnol Biochem ; 84(1): 154-158, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31794328

RESUMO

Malectin is a maltose-binding endoplasmic reticulum protein conserved in animals. In Arabidopsis thaliana, we identified four genes that encode malectin-like domain (MLD)- and leucine-rich repeat (LRR)-containing proteins (AtMLLRs): two were receptor-like proteins (AtMLLR1 and 2) and the other two were extracellular proteins (AtMLLR3 and 4). The promoter:G3GFP+promoter:GUS assay indicated the organ- and cell-specific expression of the AtMLLR2 and AtMLLR3 genes.Abbreviations: Cmr: chloramphenicol-resistance marker; G3GFP: G3 green fluorescent protein; GUS: ß-glucuronidase; KD: kinase domain; LRR: leucine-rich repeat; MLD: malectin-like domain; RLK: receptor-like kinase; SP: signal peptide; TMD: transmembrane domain; Tnos: nopaline synthase terminator.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expressão Gênica , Lectinas/genética , Proteínas de Membrana/genética , Proteínas/genética , Retículo Endoplasmático/metabolismo , Glucuronidase/química , Proteínas de Fluorescência Verde/química , Leucina/genética , Microscopia de Fluorescência , Filogenia , Plantas Geneticamente Modificadas , Domínios Proteicos/genética , Coloração e Rotulagem
7.
J Phys Chem Lett ; 10(24): 7817-7822, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31682445

RESUMO

We present the spectrum of the S1 ← S0 transition of an anionic model for the chromophore of the green fluorescent protein in vacuo at cryogenic temperatures, showing previously unresolved vibrational features, and resolving the band origin at 20 930 cm-1 (477.8 nm) with unprecedented accuracy. The vibrational spectrum establishes that the molecule is in the Z isomer at low temperature. At increased temperature, the S1 ← S0 band shifts to the red, which we tentatively attribute to emergent population of the E isomer.


Assuntos
Compostos de Benzilideno/química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Imidazóis/química , Modelos Moleculares , Temperatura Baixa , Íons/química , Isomerismo , Nitrogênio/química , Processos Fotoquímicos , Conformação Proteica , Solventes/química , Espectrometria de Fluorescência/métodos , Termodinâmica , Vibração
8.
Phys Rev Lett ; 123(12): 128101, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31633990

RESUMO

Recent experimental results have shown that enzymes can diffuse faster when they are in the presence of their reactants (substrate). This faster diffusion has been termed enhanced diffusion. Fluorescence correlation spectroscopy (FCS), which has been employed as the only method to make these measurements, relies on analyzing the fluctuations in fluorescence intensity to measure the diffusion coefficient of particles. Recently, artifacts in FCS measurements due to its sensitivity to environmental conditions have been evaluated, calling prior enhanced diffusion results into question. It behooves us to adopt complementary and direct methods to measure the mobility of enzymes. Herein, we use a technique of direct single molecule imaging to observe the diffusion of individual enzymes in solution. This technique is less sensitive to intensity fluctuations and deduces the diffusion coefficient directly based on the trajectory of the enzyme. Our measurements recapitulate that enzyme diffusion is enhanced in the presence of its substrate and find that the relative increase in diffusion of a single enzyme is even higher than those previously reported using FCS. We also use this complementary method to test if the total enzyme concentration affects the relative increase in diffusion and if the enzyme oligomerization state changes during its catalytic turnover. We find that the diffusion increase is independent of the total concentration of enzymes and the presence of substrate does not change the oligomerization state of enzymes.


Assuntos
Enzimas/metabolismo , Imagem Molecular/métodos , Difusão , Enzimas/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Cinética , Espectrometria de Fluorescência/métodos , Ureia/metabolismo , Urease/química , Urease/metabolismo
10.
Nat Biotechnol ; 37(11): 1287-1293, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31548726

RESUMO

Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.


Assuntos
Aptâmeros de Nucleotídeos/genética , RNA/genética , RNA/metabolismo , Animais , Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Humanos , Mamíferos , Microscopia de Fluorescência , Biossíntese de Proteínas , RNA/química , Transcrição Genética
11.
Biochemistry (Mosc) ; 84(8): 931-940, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522675

RESUMO

Clostridium thermocellum lichenase (endo-ß-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.


Assuntos
Clostridium thermocellum/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Domínios Proteicos , Engenharia de Proteínas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Elementos de DNA Transponíveis , Escherichia coli/citologia , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Redobramento de Proteína , Proteínas Recombinantes de Fusão , Temperatura Ambiente
12.
Biochimie ; 167: 93-105, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31560933

RESUMO

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the Kds measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots.


Assuntos
Cisteína/química , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Multimerização Proteica , Pontos Quânticos/química , Sequência de Aminoácidos , Dimerização , Ligações de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-Hélice
13.
Nanoscale ; 11(32): 15307-15311, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31386727

RESUMO

Vaccines for many important diseases remain elusive, and those for others need to be updated frequently. Vaccine efficacy has been hindered by existing sequence diversity in proteins and by newly-acquired mutations that enable escape from vaccine-induced immune responses. To address these limitations, we developed an approach for nanopatterning protein antigens that combines the site-specific incorporation of non-canonical amino acids with chemical modification to focus the immune response on conserved protein regions. We demonstrated the approach using green fluorescent protein (GFP) as a model antigen and with a promising malaria vaccine candidate, Merozoite surface protein 119 (MSP119). Immunization of mice with nanopatterned MSP119 elicited antibodies that recognized MSP119 from heterologous strains, differing in sequence at as many as 21 of 96 residues. Nanopatterning should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.


Assuntos
Antígenos/imunologia , Nanoestruturas/química , Animais , Anticorpos Antiprotozoários/imunologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/imunologia , Vacinas Antimaláricas/imunologia , Camundongos , Mutagênese , Plasmodium falciparum/metabolismo , Polietilenoglicóis/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
14.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366154

RESUMO

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.


Assuntos
Grafite/química , Proteínas de Fluorescência Verde/química , Hidrogéis/química , Proteínas Imobilizadas/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Alginatos/química , Técnicas Biossensoriais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metacrilatos/química , Nanopartículas/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química
15.
Anticancer Res ; 39(8): 4055-4060, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366487

RESUMO

BACKGROUND/AIM: Tumor-derived exosomes play important roles in tumor metastases. In this report, we observed the fate of tumor-derived exosomes in pancreatic cancer metastatic nude-mouse models using color-coded imaging. MATERIALS AND METHODS: Mia-PaCa-2 human pancreatic cancer cells expressing red fluorescent protein (RFP) were transduced by exosome-specific pCT-CD63-green fluorescent protein (GFP) and injected in the spleen of nude mice. RESULTS: Four weeks after injection of these cells into the spleen, liver metastases developed and tumor-derived exosomes were observed within the metastatic cancer cells and in Kupffer cells. Furthermore, tumor-derived exosomes diffused to bone marrow and lung cells, especially macrophages, without any metastases present. CONCLUSION: In the present study, we visualized the distribution of cancer-derived exosomes for the first time at the cellular level, in a pancreatic-cancer metastatic model.


Assuntos
Linhagem da Célula/genética , Exossomos/genética , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Pancreáticas/diagnóstico por imagem , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/química , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Proteínas Luminescentes/química , Camundongos , Metástase Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Anticancer Res ; 39(8): 4061-4064, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366488

RESUMO

BACKGROUND/AIM: Circulating tumor cells (CTCs) may have an important role in metastasis. CTC clusters, which contain fibroblasts, indicate poor prognosis. In the present study, we used our malignant lymphoma metastatic mouse model to compare the effect of a choline-deficient-diet (CDD) and the control diet (CD) on fibroblasts in CTCs. MATERIALS AND METHODS: We compared the number and morphology of CTCs in CDD and CD mice using color-coded imaging with fluorescent proteins. Malignant lymphoma EL4 cells expressing RFP were injected in the spleen of transgenic C57B/6-GFP mice, which were fed a CDD or CD. Two weeks later, we harvested and observed the number of CTCs and fibroblast-like cells both in heart blood and portal blood. Imaging of CTC morphology was performed with smeared glass slides and in culture. RESULTS AND CONCLUSION: There was no significant difference in the number of CTCs between CDD and CD mice. The number of fibroblast-like cells in the CTC microenvironment in CD mouse portal blood was significantly larger than in CDD mouse portal blood. These differences may be caused by deficiency in choline that leads to less metastasis in choline-deficient-diet-induced fatty liver.


Assuntos
Colina/metabolismo , Linfoma/sangue , Células Neoplásicas Circulantes/metabolismo , Células Estromais/metabolismo , Animais , Linhagem Celular Tumoral , Deficiência de Colina/sangue , Deficiência de Colina/genética , Deficiência de Colina/patologia , Dieta/efeitos adversos , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Fluorescência Verde/química , Humanos , Proteínas Luminescentes/química , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Células Estromais/patologia , Microambiente Tumoral/genética
17.
Anticancer Res ; 39(8): 4065-4071, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366489

RESUMO

BACKGROUND: Surgical orthotopic implantation of human colon cancer tissue to the ceca of mice has been used to mimic behavior of cancer in human patients for the development of precision cancer medicine. However, with the current method of serosal surface implantation (SSI) of pieces of human colon cancer tissue, cancer cells are exposed to the peritoneum, which can artificially increase the rate of peritoneal carcinomatosis (PC) during the disease course. The objective of the present study was to introduce a tumor-sealing method (TSM) and compare it with SSI for the ability to produce clinically-relevant metastases without artificial PC. MATERIALS AND METHODS: HCT116 colon cancer cells transfected with green fluorescence protein (GFP) were cultured and then injected into the subcutaneous layer of athymic nude mice. Subcutaneous tumors were allowed to grow sufficiently to supply adequate tumor for orthotopic implantation. For SSI, a 1 mm3-sized tumor fragment was sutured to partially torn serosa of the cecum. For TSM, the blind end of the cecum was folded over the tumor fragment and sealed with sutures. At 20 days after implantation, all mice were opened to visualize PC by intravital fluorescence imaging. At necropsy, distant metastasis was investigated using frozen section of whole blocks of organs. RESULTS: At 20 days after implantation, PC rates in the SSI group and the TSM group were 80% (12/15) and 20% (3/15), respectively (p<0.001). The liver metastasis rate was 41.7% (5/12) in the SSI group and 50% (5/10) in the TSM group (p=0.696). The lung metastasis rate was 0% (0/12) in the SSI group and 10% (1/10) in the TSM group (p=0.201). The mean survival of mice without PC on the 20th day was significantly longer than that of mice with PC on the 20th day (69.1±14.7 vs. 44.5±12.4 days, p=0.001). CONCLUSION: These results suggest that TSM might be a more patient-like and useful method as a model of metastatic colon cancer than SSI.


Assuntos
Carcinogênese/genética , Neoplasias do Colo/patologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/patologia , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/cirurgia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/química , Células HCT116 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Metástase Neoplásica , Transplante de Neoplasias/métodos , Imagem Óptica , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461959

RESUMO

Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors.


Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/química , Absorção de Radiação , Animais , Genes Reporter , Proteínas de Fluorescência Verde/classificação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Phys Chem Chem Phys ; 21(35): 18988-18998, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31464320

RESUMO

Computational approaches have to date failed to fully capture the large (about 0.4 eV) excitation energy tuning displayed by the nearly identical anionic chromophore in different green fluorescent protein (GFP) variants. Here, we present a thorough comparative study of a set of proteins in this sub-family, including the most red- (phiYFP) and blue-shifted (mTFP0.7) ones. We employ a classical polarisable embedding through induced dipoles and combine it with time-dependent density functional theory and multireference perturbation theory in order to capture both state-specific induction contributions and the coupling of the polarisation of the protein to the chromophore transition density. The obtained results show that only upon inclusion of both these two effects generated by the mutual polarisation between the chromophore and the protein can the full spectral tuning be replicated. We finally discuss how this mutual polarisation affects the correlation between excitation energies, dipole moment variation, and molecular electrostatic field.


Assuntos
Cor , Polarização de Fluorescência , Proteínas de Fluorescência Verde/química , Eletricidade Estática
20.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
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