Optimization of Protein-Protein Interaction Measurements for Drug Discovery Using AFM Force Spectroscopy

Increasingly targeted in drug discovery, protein-protein interactions challenge current high throughput screening technologies in the pharmaceutical industry. Developing an effective and efficient method for screening small molecules or compounds is critical to accelerate the discovery of ligands for enzymes, receptors and other pharmaceutical targets. Here, we report developments of methods to increase the signal-to-noise ratio (SNR) for screening protein-protein interactions using atomic force microscopy (AFM) force spectroscopy. We have demonstrated the effectiveness of these developments on detecting the binding process between focal adhesion kinases (FAK) with protein kinase B (Akt1), which is a target for potential cancer drugs. These developments include optimized probe and substrate functionalization processes and redesigned probe-substrate contact regimes. Furthermore, a statistical-based data processing method was developed to enhance the contrast of the experimental data. Collectively, these results demonstrate the potential of the AFM force spectroscopy in automating drug screening with high throughput

Bacteria in the lakes of the Tibetan Plateau and polar regions

The Tibetan Plateau, also termed ‘the Third Pole’ harbors the largest number of high-altitude lakes in the world. Due to the presence of extreme conditions such as low temperature and oligotrophy, the lakes of the Tibetan Plateau share environmental features in common with lakes in the polar regions. However, the extent to which these environments are analogous, or indeed whether they harbor similar microbial communities or a high level of endemic species is poorly understood. Here we compared high-throughput 16S rRNA gene sequencing data from the lakes of the three different regions in order to characterize their taxonomic diversity, the community composition and biogeography. Our results showed despite the similarity in environmental conditions, the spatial distribution of the bacterial communities was distinct with only 3.1% of all operational taxonomic units (OTUs) being present in all three regions (although these OTUs did account for a considerable proportion of the total sequences, 36.4%). Sequences belonging to Burkholderiales and Actinomycetales dominated the shared OTUs across all three regions. Scale dependent distance decay patterns provided evidence of dispersal limitation. Climatic variables and dispersal limitation were apparently both important in controlling the spatial distribution of bacterial communities across regions. This work expands our understanding of the diversity and biogeography of lake bacterial communities across the Tibetan Plateau and provides insights into how they compare to those of the Antarctic and Arctic

Measurement of cationic and intracellular modulation of integrin binding affinity by AFM-Based nanorobot

Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg2+ to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca2+ virtually abolished the RGD-membrane specific interactions and blocked the Mg2+ effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study. © 2013 Biophysical Society.Link_to_subscribed_fulltex