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The Contribution of Methionine to the Stability of the Escherichia Coli Metniq ABC Transporter - Substrate Binding Protein Complex

Nguyen, Phong T. (2016) The Contribution of Methionine to the Stability of the Escherichia Coli Metniq ABC Transporter - Substrate Binding Protein Complex. Biophysical Journal, 110 (3). 140A. ISSN 0006-3495. http://resolver.caltech.edu/CaltechAUTHORS:20160606-075145880

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Abstract

Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250-253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ∼40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.bpj.2015.11.795DOIArticle
http://www.sciencedirect.com/science/article/pii/S0006349515019785PublisherArticle
Additional Information:© 2016 Biophysical Society. Published by Elsevier Inc.
Record Number:CaltechAUTHORS:20160606-075145880
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160606-075145880
Official Citation:Phong T. Nguyen, The Contribution of Methionine to the Stability of the Escherichia Coli Metniq ABC Transporter - Substrate Binding Protein Complex, Biophysical Journal, Volume 110, Issue 3, Supplement 1, 16 February 2016, Page 140a, ISSN 0006-3495, http://dx.doi.org/10.1016/j.bpj.2015.11.795. (http://www.sciencedirect.com/science/article/pii/S0006349515019785)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67669
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 Jun 2016 16:24
Last Modified:06 Jun 2016 16:24

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