Biology subjects

Akpinar, B.

Publications and source records attributed to Akpinar, B..

2 recordsLinked to original sources

bioRxiv2019

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working at biologically relevant protein concentrations, where protein may be present at large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113 / PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show that this selective adsorption extends to DNA-protein complexes, through the use of biotinylated DNA and streptavidin, and demonstrate that DNA-bound streptavidin can be unambiguously distinguished by in-liquid AFM in spite of an excess of unbound streptavidin in solution.

biophysics↗

bioRxiv2019

Quantification of biomolecular dynamics inside real and synthetic nuclear pore complexes using time-resolved atomic force microscopy

Over the past decades, atomic force microscopy (AFM) has emerged as an increasingly powerful tool to study the dynamics of biomolecules at nanometre length scales. However, the more stochastic the nature of such biomolecular dynamics, the harder it becomes to distinguish them from AFM measurement noise. Rapid, stochastic dynamics are inherent to biological systems comprising intrinsically disordered proteins. One role of such proteins is in the formation of the transport barrier of the nuclear pore complex (NPC): the selective gateway for macromolecular traffic entering or exiting the nucleus. Here, we use AFM to observe the dynamics of intrinsically disordered proteins from two systems: the transport barrier of native NPCs, and the transport barrier of a mimetic NPC made using a DNA origami scaffold. Analysing data recorded with 50-200 ms temporal resolution, we highlight the importance of drift correction and appropriate baseline measurements in such experiments. In addition, we describe an auto-correlation analysis to quantify time scales of observed dynamics and to assess their veracity -- an analysis protocol that lends itself to the quantification of stochastic fluctuations in other biomolecular systems. The results reveal the surprisingly slow rate of stochastic, collective transitions inside mimetic NPCs, highlighting the importance of FG-nup cohesive interactions.

biophysics↗