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Molecular movements of neural transporters unveiled
Date:4/25/2011

method does not simply generate information about the average movements of a collection of proteins. "Applying single-molecule imaging to the transporter gave us a unique view of the dynamics enabling the function of the transporters, because it allowed us to look at individual molecules and watch their movements in real time rather than time and ensemble averages," Dr. Blanchard says.

Last year, the researchers used smFRET to collect the first such single-molecule data for a membrane protein, and these results were also published in Nature. In their most recent experiments, they used the technique to monitor changes in LeuT conformation and dynamics by labeling moving parts of the protein with fluorescent dyes that emit distinct amounts of light when the distance separating them changes. As the transporter protein moves during function, time-dependent changes in distance between the fluorophores could be directly imaged to extract the first quantitative insights into the motions underpinning the transport mechanism.

Using powerful computational simulations, the researchers had predicted such movements through previous studies aimed at understanding how the transported molecule changes the conformation of LeuT. The new experiments demonstrated that alanine binding to LeuT increased the rate of the transporter's flickering between two conformations: facing outward, as if ready to accept substrates from outside the cell ("inward-closed"), and facing inward, as if releasing its contents into the cell ("inward-open"). How the presence of sodium affects the transporter's response to the binding of the transported substrate, alanine, was also revealed from these experiments: Sodium was essential for the alanine-enhanced dynamics. Surprisingly, alanine did not alter the total amount of time spent in either the open or closed state.

By contrast, the binding of sodium ions alone, without alanine, was found to decrease the transition rate between o
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Contact: Andrew Klein
ank2017@med.cornell.edu
212-821-0560
New York- Presbyterian Hospital/Weill Cornell Medical Center/Weill Cornell Medical College
Source:Eurekalert

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