Abstract
Protein kinases play central role in cellular regulation by catalyzing the phosphorylation of target proteins involved in complex physiological processes, achieved by their structural flexibility switching between active state and inactive states. Quantitative characterization of kinase conformational dynamics is important yet challenging. Nanopore analysis is emerging as an attractive single-molecule tool for probing protein conformational dynamics due to several advantages, such as label-free, and being able to detect structural dynamics in a wide range of timescales (μs∼mins).
Highlights
Protein kinases are attractive drug targets because they play a central role in regulating the majority of cellular pathways by catalyzing the phosphorylation of target proteins involved in complex physiological processes.[1]
Abl kinase has a conserved catalytic kinase domain, consisting of a N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe) that are linked by a flexible hinge
Conformational states of Abl kinase domain are usually classified according to the arrangement of four key elements including the activation loop (A-loop), the Asp-Phe-Gly motif(DFG-motif), the glycine rich loop (G-loop), and the αC-helix (Fig. 1a).[11, 12]
Summary
Protein kinases are attractive drug targets because they play a central role in regulating the majority of cellular pathways by catalyzing the phosphorylation of target proteins involved in complex physiological processes.[1]. Apart from the general strengths of single-molecule techniques such as the ability to access conformational heterogeneity, transient or sparsely populated intermediates, and sequential steps of enzyme’s catalytic cycle 28-32, nanopore tweezers own some unique advantages including being label-free and capable of detecting structural dynamics in a wide range of timescales (μs~mins).[33] Previous works showed that natively folded proteins 26, 34-37 can be reversibly trapped with Cytolysin A (ClyA) nanopore variants to reveal their ligand bound and catalytic states. In this study we used ClyA nanopore tweezers to track structural dynamics of the Abl kinase domain
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