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1.
J Fluoresc ; 30(3): 703-715, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32385659

RESUMO

mEos3.2 is a photoconvertible fluorescence protein with comparatively low brightness, which limits its application in live Super resolution microscopy. To address this issue, we have used semi-rational protein engineering to develop mEosBrite, a new class of improved brightness variants. The improvement in the brightness was confirmed by expression in E.coli as well as mammalian cell lines. Furthermore, biophysical characterization suggests that all the three mEosBrite variant proteins display higher quantum yield, truly monomeric form, less cytotoxicity and lower protein aggregation as compared to the wild type mEos3.2 protein. Most importantly, because of their high photoconversion efficiency mEosBrite variants could be an excellent tool for single-molecule and intensity fluctuation based super-resolution microscopy.


Assuntos
Proteínas Luminescentes/química , Engenharia de Proteínas , Escherichia coli/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Modelos Moleculares , Imagem Óptica
2.
Protein Sci ; 30(9): 1958-1973, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34191384

RESUMO

T-cell co-stimulation through CD28/CTLA4:B7-1/B7-2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA-approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7-2 on the cell surface. Here, we describe the structure of the IgV domain of B7-2 and its cryptic association into 1D arrays that appear to represent the pre-signaling state of B7-2 on the cell membrane. Super-resolution microscopy experiments on heterologous cells expressing B7-2 and B7-1 suggest, B7-2 form relatively elongated and larger clusters compared to B7-1. The sequence and structural comparison of other B7 family members, B7-1:CTLA4 and B7-2:CTLA-4 complex structures, support our view that the observed B7-2 1D zipper array is physiologically important. This observed 1D zipper-like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.


Assuntos
Antígeno B7-1/química , Antígeno B7-2/química , Antígenos CD28/química , Antígeno CTLA-4/química , Sequência de Aminoácidos , Animais , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Sítios de Ligação , Antígenos CD28/genética , Antígenos CD28/metabolismo , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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