Assuntos
Transferência Ressonante de Energia de Fluorescência , Nuclease do Micrococo/metabolismo , Staphylococcus/enzimologia , Substituição de Aminoácidos , Dicroísmo Circular , Cinética , Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Técnicas Analíticas Microfluídicas , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Periplásmicas/metabolismo , Porinas/metabolismo , Serina Endopeptidases/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência , Proteínas de Choque Térmico/química , Cinética , Chaperonas Moleculares/química , Peptidilprolil Isomerase/química , Periplasma/metabolismo , Proteínas Periplásmicas/química , Porinas/química , Transporte Proteico , Serina Endopeptidases/químicaRESUMO
It has been a long-standing challenge in bioassay using aptamers and gold nanoparticles to detect disease-related proteins and other substance directly in complex biological samples such as serum. Here we propose a progressive dilution (PD) method to achieve simultaneous qualitative and quantitative analysis of proteins in blood serum without pretreatment of the sample. Above the detection limit, PD has unlimited dynamic range. We demonstrate the PD strategy through the detection of thrombin in fetal bovine serum using the quenching of fluorescence by gold nanoparticles.