The CLC 'Cl À channel' family consists of both Cl À /H þ antiporters and Cl À channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl À permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent 19 F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl À permeation pathway and show the usefulness of 19 F NMR for studying conformational changes in membrane proteins of known structure.

changes induced were large but two intracellular Cl induced the largest conformational change, repelling the side chain of E148 against the external channel wall. This distortion produced a pathway that had an area 2.4 times bigger than the one seen with COO À and no Cl. We anticipate that this larger pathway will allow Cl conduction easily. Our results imply that the combine actions of Cl and protonation of the E148 lateral chain are necessary to open the pore. Finally, the energy barriers that Cl faces during conduction strongly depend on structure, relative orientation, and chemical composition of the pore entryway. Supported by CONACyT grants 45928 (RG) and 79897 (JA).

Notwithstanding the “one-module-one-elongation-cycle” paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as “stuttering”, have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all trans-configured triene moiety in the polyketide product. Here we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the “split-and-stuttering” module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.TOC/Abstract Graphic

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