| This thesis describes three investigations encompassing protein dynamics, protein structure, and NMR methods development, representing three major areas of modern biological NMR spectroscopy. Cadherins are cell-surface molecules which mediate cellular adhesion, through dimerization of cadherin N-terminal extracellular (EC1) domains presented from apposed cells. Exchange of N-terminal beta-strands between EC1 domains leads to symmetrical insertion of conserved tryptophan side chains from one monomer into a hydrophobic acceptor pocket of the partner molecule. In Chapters 2-5, NMR spectroscopy is used to characterize monomer and dimer states of the mouse Type II cadherin-8 EC1 domain. Limited picosecond-to-nanosecond timescale dynamics of the tryptophan indole side chains for both monomer and dimer states are consistent with well-ordered packing of the N-terminal beta-strands intramolecularly and intermolecularly, respectively. However, pronounced microsecond-to-millisecond timescale dynamics of the side chains are observed only for the monomer state, suggesting that monomer, but not dimer, species sample configurations in which the indole moieties are exposed. The results support a selected-fit mechanism for EC1 domain dimerization, where collisions between strand-exposed monomers lead to formation of the strand-swapped dimer state.; Voltage-gated sodium channels are critical components of the action potential in excitable cells, and disruption of their function is associated with arrhythmogenic and epileptogenic syndromes. In Chapters 6-9 the Nav1.2 voltage-gated sodium channel C-terminal domain (1777-1882) solution structure is determined at pH 7.4, 290 K. The structure is homologous to a paired EF-Hand domain, although the EF-Hand motifs are not functional calcium binding sites, as evidenced by chemical shift perturbation analysis. Similar calcium binding properties are observed for the C-terminal domain of the homologous Nav1.5 isoform (1773-1878), which is expected to adopt a similar topological fold on the basis of secondary structure predictions. By analogy to target binding by calmodulin, and the previous demonstration of interaction between the Na v1.5 C-terminal domain a downstream calmodulin target binding site, a model for sodium channel inactivation in pathological states is hypothesized.; In Chapters 10-12 spin relaxation induced by chemical exchange is examined theoretically. Analytic expressions for the nuclear magnetic spin relaxation rate constant for magnetization spin-locked in the rotating reference frame under an applied radio-frequency field, R1rho , are obtained for two-site chemical exchange. The theoretical approach is motivated by Laguerre's Method and obtains R1rho as the root of a (p1, q 1) Pade approximant. The general formula for R 1rho obtained by this approach is substantially simpler than existing expressions and is equally or slightly more accurate, in most cases. In addition, particular solutions for the R1rho rate constant are presented for two special cases: equal populations of the two exchanging sites, or placement of the radio-frequency carrier at the average resonance frequency of the two sites. The solutions are exact when the R 1 and R2 relaxation rate constants are identical, and nearly exact under realistic experimental conditions. |
