Members of the CLC family orchestrate the movements of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. In order to better understand the mechanisms underlying chloride transport by CLC proteins, this study will examine activity-associated conformational changes occurring in the E. coli CLC homolog, CLC-ec1, for which an x-ray crystallographic structure has been solved. Because of mechanistic and structural similarities among CLC family members, information gained in this study will be relevant to other CLCs, including those responsible for a variety of human diseases. To facilitate transport, CLC-ec1 must cycle through several conformations. Since the crystal structure of CLC-ec1 shows only a static picture, it cannot offer information concerning protein movement. In this study, changes in CLC-ec1 conformation will be assessed by fluorine-labeling specific residues and monitoring their movement via changes in their 19F resonance. When evaluated in light of the crystal structure of CLC-ec1, this information will allow us to determine which parts of the protein move in order to catalyze transport. Additionally, paramagnetic probes will be used to assess the distance between labeled residues and the aqueous environment, allowing for measurement of the size and degree of opening of vestibules contained within CLC-ec1. Kinetics of CLC-ec1 movement will be examined by determining the temperature-dependence of 19F resonance changes. Finally, mutant forms of CLC-ec1 that are known to have altered function will be examined to determine the effect of these mutations on conformational changes. Together, these results will allow us to develop models describing how CLC proteins move as they catalyze ion transport.
Defects in CLC chloride transport proteins can lead to a variety of human diseases, including epilepsy, myotonia, Barrier's syndrome, osteoporosis, and Dent's disease. This study will examine the molecular mechanisms of CLC-mediated chloride transport, which is essential for the development and application of therapies to treat these diseases.
| Elvington, Shelley M; Liu, Corey W; Maduke, Merritt C (2009) Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR. EMBO J 28:3090-102 |
