Protein kinases play central roles in cellular regulation by catalyzing the phosphorylation of target proteins. Kinases have inherent structural flexibility allowing them to switch between active and inactive states. Quantitative characterization of kinase conformational dynamics is challenging. Here, we use nanopore tweezers to assess the conformational dynamics of Abl kinase domain, which is shown to interconvert between two major conformational states where one conformation comprises three sub-states. Analysis of kinase-substrate and kinase-inhibitor interactions uncovers the functional roles of relevant states and enables the elucidation of the mechanism underlying the catalytic deficiency of an inactive Abl mutant G321V. Furthermore, we obtain the energy landscape of Abl kinase by quantifying the population and transition rates of the conformational states. These results extend the view on the dynamic nature of Abl kinase and suggest nanopore tweezers can be used as an efficient tool for other members of the human kinome.
Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate singlemolecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.
Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.
Latinas are often more affected by HIV due to their socio-economic and demographic profiles and are also less likely to receive proper mental health care. Latina immigrants are often even more vulnerable due to socio-economic and cultural factors that place them at higher risk. The current study seeks to examine the association between depression and risky sexual behaviors among adult Latina immigrants from a farm working community in South Miami-Dade County, (Florida, USA). Cross-sectional secondary data analysis was used for responses from a community-based participatory research (CBPR) study. Out of 234 Latina immigrants, 15% reported being depressed and 80% were reported as having engaged in risky sexual behavior. Although no association was found between depression and high-risk sexual behavior, significant secondary findings present associations between risky sexual behavior and low sexual relationship power, interpersonal violence, and relationship status. Implications for future research on depression and risky sexual behaviors among this population are discussed.
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