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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.