1. Structural and dynamic determinants of ion channel assembly by scaffolding proteins.
Ion transport across cell membranes is essential for cell survival. The cystic fibrosis transmembrane conductance regulator (CFTR) is the most significant ion channel in several epithelial tissues, particularly in the lungs and colon, where CFTR is responsible for salt and fluid transport. Defects in the CFTR gene lead to cystic fibrosis, which is the most common genetic disease among Caucasians. In cystic fibrosis patients, the lack of ion transport by CFTR causes the lungs to secrete thick mucus that blocks the airways, causing a bacterial buildup that triggers lethal inflammation.
Two adapter proteins—Na+/H+ exchanger regulator factor (NHERF) and ezrin—enhance the cell surface
expression of CFTR, organize the macromolecular interactions of CFTR with other signaling proteins for
efficient signal transduction, and modulate the strength of ion transport. Recently, our laboratory provided
the first biochemical evidence that binding of ezrin to NHERF changes the stoichiometry of interaction of
NHERF with CFTR (1), Figure 1. The long-term goal is to determine how macromolecular interactions
influence CFTR assembly and function, allowing the structure-based design of new therapeutics for CF.
2. Protein domain motion probed by neutron scattering
Another research direction is to study protein dynamics by various means, most notably neutron
scattering. We have applied quasielastic neutron scattering for the study of protein dynamics in solution
(2,3). Recently, we have used neutron spin echo spectroscopy (NSE) to reveal long-range coupled,
overdamped domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase) on
nanosecond time scales (3). This study revealed for the first time long-range coupled correlated domain
motion within a protein. Such long-range coupling is significant biologically, for it allows DNA replication
and repair to occur with great accuracy. Specifcally, this protein utilizes conformational changes over 70 Å
to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. NSE spectroscopy
determines the domain mobility tensor, which in turn reveals the degree of dynamical coupling between
domains separated by 70 Å. Our long-term goal is to determine the extent to which coupled protein domain
motion influences biological function and thus the fidelity of DNA replication.
References