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Abstract: Transmembrane signaling events that propagate through receptors and transporters have critical roles in cellular function and regulation. In the Escherichia coli vitamin B12 transporter, BtuB, substrate binding to the extracellular surface of the protein triggers the unfolding of an energy coupling motif at the periplasmic surface. Here, the molecular interactions mediating this substrate-dependent transmembrane signaling event were investigated in a novel way by combining a two mutant cycle analysis with site-directed spin labeling (SDSL). SDSL was used to monitor the unfolding and conformational equilibrium of the energy-coupling motif, and a thermodynamic two-mutant cycle analysis was used to estimate pair-wise interaction free energies for a pair of charged residues (D316 and R14) within the protein interior. The data indicate that D316 and R14 are critical to this structural transition. Substrate binding is shown to reduce the interaction free energy between these residues, thereby triggering the unfolding of the energy coupling motif of this membrane transporter. The result indicates that SDSL when used in combination with a mutant cycle analysis provides an approach to examine the molecular interactions mediating signaling events in membrane proteins. [Copyright &y& Elsevier]

Core binding factors (CBFs) are heterodimeric transcription factors consisting of a DNA-binding CBFα subunit and non-DNA-binding CBFβ subunit. DNA binding and heterodimerization is mediated by a single domain in the CBFα subunit called the Runt domain, while sequences flanking the Runt domain confer other biochemical activities such as transactivation. On the other hand, the heterodimerization domain in CBFβ is the only functional domain that has been identified in this subunit. The biophysical properties of the Runt domain and the CBFβ heterodimerization domain, and the structures of the isolated domains as well as of the Runt domain-DNA, Runt domain-CBFβ, and ternary Runt domain-CBFβ-DNA complexes, have been characterized over the past several years, and are summarized in this review. [Copyright &y& Elsevier]