Dance Lessons for Proteins: The Dynamics and Thermodynamics of a Sodium/Aspartate Symporter

Chapter
Part of the Springer Series in Biophysics book series (BIOPHYSICS, volume 17)

Abstract

Secondary active transporters harvest the energy of the ionic gradients to drive concentrative uptake of their substrates. This process entails a series of protein conformational transitions that are coupled to binding and unbinding of ions and substrates on the extracellular and intracellular sides of the membrane. Over the last decade, crystallography has provided a growing number of structural snapshots of the transport cycle for several ion-driven transporters. Already these structures, although intrinsically static, have revealed a remarkable plasticity encoded in the architecture of these proteins. Because internal dynamics is an essential feature of transporters, it is necessary to complement crystallographic studies with other techniques that provide information on the ensemble properties of these proteins as well as on the conformational dynamics of individual molecules. Here, we will discuss the emerging approaches to obtain thermodynamic and dynamic information on transporters using a sodium/aspartate symporter from Pyrococcus horikoshii, GltPh, as a model system. GltPh is a bacterial homologue of the mammalian glutamate transporters, for which crystal structures of several states have been determined, providing a framework for further mechanistic studies. We will discuss how within this system the equilibrium and kinetic studies based on the isothermal titration calorimetry, fluorescence, and electron paramagnetic resonance spectroscopy inform us on the energetic relationship between the key functional states, and mechanisms of coupling between transport cycle and ionic gradients. We will further describe how single molecule studies open doors to a detailed characterization of the timing and order of the conformational transitions underlying transport processes.

Keywords

Glutamate transporters Sodium symporters Functional dynamics Anion permeation Substrate binding Alternating access Secondary transport Conformational changes 

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© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Weill Cornell Medical CollegeNew YorkUSA

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