Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Poster Abstracts

18-POS Board 18 Signaling-related Structural Changes of Chemoreceptor Nano-Arrays Elizabeth R. Haglin , Lynmarie K. Thompson. University of Massachusetts Amherst, Amherst, MA, USA.

In bacteria, 200-nm extended networks of membrane protein nano-arrays cooperatively sense and adapt to environmental stimuli in order to direct swimming patterns. Upon ligand binding to the chemoreceptors, a small 2Å downward piston motion is able to promote inactivation of the histidine kinase, CheA more than 250Å away. Understanding the mechanism of this phenomenon could lead to development of novel antibiotics capable of disrupting bacterial chemotaxis, thus inducing nutrient starvation and death. We assemble native-like functional chemoreceptor arrays by binding a His-tagged cytoplasmic fragment of the Asp receptor to templating vesicles in the presence of the other protein components, a coupling protein CheW and kinase CheA. Electron cryotomography (ECT) demonstrates that these in vitro arrays consist of extended hexagonal assemblies with each hexagon formed by six trimers of receptor dimers, with the same 12-nm spacing seen for intact receptors in cells. Assembly density has been shown to alter kinase and methylation activities in an inverse fashion, analogous to the physiological signaling states. We hypothesize that the kinase-off/methylation-on signaling state has an expanded membrane-proximal region relative to the kinase-on/methylation-off signaling state. To test this hypothesis, we are investigating the membrane-proximal area of each signaling state by measuring protein binding and activities for arrays assembled on vesicles of known surface area. Preliminary results indicate that greater crowding is only possible by restricting the surface area during, rather than after array assembly. This suggests the kinase-on array is highly stable and restricts access of additional protein into the array. For example, sterically restricting access of CheR to the receptor methylation sites is a potential mechanism for controlling methylation activity. Concurrently, we are using solid state NMR to measure dimer-dimer distances to characterize the trimer of dimers structure and quantify the proposed signaling-related receptor expansion.

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