POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
POSTERS - BLAST X - University of Utah
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
<strong>BLAST</strong> X Poster #67<br />
NEW REPORTER RESIDUES OF TRIMER FORMATION BY ESCHERICHIA COLI MCPs<br />
Diego A. Massazza 1 , John S. Parkinson 2 , Claudia A. Studdert 1<br />
1 Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Argentina<br />
2 Biology Department, <strong>University</strong> <strong>of</strong> <strong>Utah</strong><br />
It is currently accepted that E. coli chemoreceptors are organized in vivo in trimer-<strong>of</strong>dimer<br />
arrangements. Using receptors bearing a cysteine reporter residue near the trimer axis,<br />
we previously demonstrated efficient formation <strong>of</strong> 2- and 3-subunit crosslinking products upon<br />
treatment <strong>of</strong> intact cells with the trifunctional maleimide reagent TMEA. In this work, we<br />
assessed the in vivo crosslinking behavior <strong>of</strong> receptors bearing cysteine reporters at different<br />
positions along their cytoplasmic domains.<br />
We found that the formation <strong>of</strong> 3-subunit crosslinking products declined with increasing<br />
distance <strong>of</strong> the reporter from the cytoplasmic tip. This result suggests that the in vivo<br />
arrangement <strong>of</strong> the trimer <strong>of</strong> dimers resembles that observed in the crystal structure <strong>of</strong> the Tsr<br />
cytoplasmic domain, in which the dimers contact one another at the tip and splay apart in the<br />
regions that are farther from the tip.<br />
We also found that stimulation with repellents immediately before the TMEA treatment<br />
caused a slight but reproducible increase in crosslinking efficiency. This behavior is consistent<br />
with previous observations suggesting that receptor dimers within the trimer move closer<br />
together after repellent stimuli.<br />
In cells co-expressing different marked receptors, we analyzed the formation <strong>of</strong> mixed<br />
crosslinking products. When the cysteine reporters were located the same distance from the tip,<br />
mixed crosslinking products formed with high efficiency. Mixed products did not form between<br />
reporters at different distances from the tip. This result suggests that there are no significant<br />
vertical displacements within the trimer <strong>of</strong> dimers, nor sufficient flexibility between the receptors<br />
to get crosslinking between reporters at diferent levels in the dimers.<br />
118