The broad, long-term objectives of this application are to understand the structure and mechanism of the lactose permease of Escherichia coli as a paradigm for secondary transport proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. Since remarkably few residues play a critical role in a mechanism that involves widespread conformational changes, it seems likely that permease turnover involves relatively simple chemistry coupled to rigid body movements of the 12 transmembrane helices that compose the molecule. Thus, delineation of helix packing and localization of the substrate translocation pathway are essential for determining the transport mechanism, and the lactose permease is uniquely suited to this enterprise. the proposed specific aims are based on newly developed techniques that combine protein engineering and various biophysical and biochemical methods which are capable of providing information at near atomic-level resolution. The overall packing of the 12 transmembrane helices in the permease will be determined primarily by measuring distances between engineered metal binding sites and site-directed nitroxide spin labeled single-Cys residues using electron parmagnetic resonance (EPR). The conclusions derived from metal-spin label distance measurements will then be confirmed and extended by placing single or paired Cys residues in neighboring helices and studying site-directed excimer fluorescence, spin-spin interactions and peptide bond cleavage with o-phenanthroline-Cu. Another approach will involve the placement of paired His residues in neighboring helices and studying Mn2+ binding by EPR. Finally, fluorescence energy transfer will be applied to permease mutants containing a single anilinonaphthalene sulfonate-labeled Cys residue and a single Trp residue in a background devoid of other Cys and Trp residues. The substrate translocation pathway will be localized with high-affinity spin-labeled galactosides and metal-spin label distance measurements utilizing EPR. Subsequently, the pathway will be studied more precisely by studying spin-spin interactions and quenching of fluorescence by spin-labeled galactosides in permease constructs containing single spin- or fluorescent-labeled Cys residues. Secondary active transport proteins are involved in a variety of human diseases, as well as resistance to certain antibiotics and the mechanism of action of certain pharmacological agents. Many of these transporters appear to have similar secondary structures based on gene sequencing and hydropathy profiling and it seems likely that the tertiary structure and mechanism of these proteins have been conserved. Therefore, studies on bacterial transporters which are much easier to manipulate than their eukaryotic counterparts have important relevance to the analogous proteins in higher order systems, particularly with respect to the development of new approaches to structure-function relationships.
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| Smirnova, Irina; Kasho, Vladimir; Jiang, Xiaoxu et al. (2015) Transient conformers of LacY are trapped by nanobodies. Proc Natl Acad Sci U S A 112:13839-44 |
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