Dynamics of the Lactose Permease of Escherichia coli

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DESCRIPTION (provided by applicant): Ion-gradient coupled active transport is a biological phenomenon that plays important roles in health and disease. For a number of years, this laboratory has focused on the lactose permease of Escherichia coli (LacY) as a paradigm for this class of transport proteins. In order to understand the transport mechanism in greater detail, structure at the atomic level, as well as time-resolved dynamic information, is required. Recently, an x-ray structure of the inward-facing conformation of LacY, a member of the Major Facilitator Super-family containing over 3000 members, was solved at about 3.5 A. This very hydrophobic protein is composed of two symmetrical 6-helix bundles with a huge, hydrophilic internal cavity that contains the sugar binding site and is open to the cytoplasm only. At the same time, financed in part by DK 51131, the Pi's laboratory has also constructed and characterized a library of single-Cys mutants at each position of LacY, as well as other site-directed mutants, and a battery of widely used site-directed techniques that can be easily applied to dynamic studies. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism has been proposed in which the hydrophilic cavity containing the binding site is alternatively accessible to either side of the membrane. Moreover, the proton electrochemical gradient has been shown to drive sugar accumulation against a concentration gradient by accelerating a rate-limiting step (dissociation of the proton) rather than changing the affinity of the binding site on either side of the membrane. Thus, the progress achieved places this laboratory in a unique position to study the detailed mechanism of transport catalyzed by LacY. In this proposal, we will use rationally designed mutants based on the x-ray structure of LacY in conjunction with various biochemical and biophysical techniques at hand to obtain time-resolved data that will yield dynamic information regarding the conformational states of LacY and their interconversion during ligand binding and turnover.