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0344411429
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note
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5) was expressed from pCK2 (54) and mutated with QuikChange (Stratagene). The cysteine-containing aspartate receptor was expressed, purified, reacted with the methanethiosulfonate spin label, and reconstituted into E. coli membranes as described (9).
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32
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0345706019
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note
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In order to verify that the spin-labeled aspartate receptors were still functional, an in vitro phosphorylation assay was carried out on labeled, reconstituted aspartate receptor mixed with purified CheW, CheA, and CheY, as described (9). AR 10-207, AR 10-210, and AR 10-215 decreased the amount of phospho-CheY in response to aspartate similarly to wild-type aspartate receptor, suggesting that spin labeling did not substantially alter the function of these receptors.
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33
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0345274456
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note
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EPR spectra were collected with a Bruker ESP 300E X-band spectrometer equipped with a loop-gap resonator (Medical Advances). The microwave power was 8 μW and the modulation amplitude was 2 G. The protein concentration ranged from 50 to 150 μM; the concentration of aspartate was 8 mM. Addition of the amino acid serine to the same concentration as aspartate did not result in any spectral changes.
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35
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0345706018
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note
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Detergent-soluble spin-labeled aspartate receptor was mixed with an equimolar amount of unlabeled, detergent-soluble wild-type aspartate receptor. After time for subunit exchange (25), the samples were reconstituted into E. coll membranes as described (9). The comparison of the amount of protein with the spin concentration revealed that the cysteine at position 10 is approximately 50% labeled, whereas all positions on TM2 were 100% labeled. The spectral contribution from the noninteracting species as a result of incomplete labeling and unidentified background labeling (9) is subtracted from the EPR spectra.
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-
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36
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0345274453
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note
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-3 dependence on the interspin distance R; the shorter R is, the more broadened the EPR spectrum. Recent developments of an EPR spectroscopic ruler and the Fourier spectral analysis method (4) allow us to accurately determine R from the analysis of the EED-broadened EPR spectrum.
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41
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0345706017
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note
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Purified CheW and CheA (9) were each added to reconstituted spin-labeled aspartate receptor, such that there was an equimolar amount of each. These were allowed to associate for at least 2 hours at room temperature, and EPR spectra were collected as for the receptor alone. Neither CheA or CheW added alone (at molar concentrations equal to or twice that of the receptor) caused spectral changes, suggesting that the changes are specific to the ternary complex. Similar CheW-CheA effects were seen with all spinlabeled aspartate receptors examined (spin labels at receptor residues 10-205, 10-211, 10-214, and 2-211).
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42
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0026808410
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0344842982
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note
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The detergent-soluble spin-labeled aspartate receptor was mixed with an equimolar amount of unlabeled detergent-soluble wild-type aspartate receptor, the subunits allowed to exchange, and the samples reconstituted into E. coli membranes. After the reconstitution, purified CheA and CheW were added and spectra collected as in (41).
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50
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0344842981
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55
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0344411423
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note
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Supported by NIH grant DK09765 and the William Keck Foundation (D.E.K.), NIH grant GM51290 and a Searle Scholarship (Y.-K.S.), and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (K.M.O.). We thank J. Falke for the coordinates of the modeled transmembrane domain aspartate receptor and S. Doyle, S. Marqusee, E. Strauss, and Z. Zhu for comments on the manuscript.
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