Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate. This is certainly the case for many pharmaceutical compounds that are absorbed in the gut or cross the blood brain barrier through endogenous transporters. Docking studies on the bacterial sugar transporter vSGLT reveal that many highly toxic compounds are compatible with binding to the orthosteric site, further motivating the selective pressure for additional modes of selectivity. Motivated by recent work in which we observed failed substrate delivery in a molecular dynamics simulation where the energized ion still goes down its concentration gradient, we hypothesize that some transporters evolved to harness this 'slip' mechanism to increase substrate selectivity and reduce the uptake of toxic molecules. Here, we test this idea by constructing and exploring a kinetic transport model that includes a slip pathway. While slip reduces the overall productive flux, when coupled with a second toxic molecule that is more prone to slippage, the overall substrate selectivity dramatically increases, suppressing the accumulation of the incorrect compound. We show that the mathematical framework for increased substrate selectivity in our model is analogous to the classic proofreading mechanism originally proposed for tRNA synthase; however, because the transport cycle is reversible we identified conditions in which the selectivity is essentially infinite and incorrect substrates are exported from the cell in a 'detoxification' mode. The cellular consequences of proofreading and membrane slippage are discussed as well as the impact on future drug development.
A comparative study of the impact of small, medium-sized, and bulky α,β-dehydroamino acids (ΔAAs) on the structure and stability of Balaram’s incipient 310-helical peptide (1) is reported. Replacement of the N-terminal Aib residue of 1 with a ΔAA afforded peptides 2a–c that maintained the 310-helical shape of 1. In contrast, installation of a ΔAA in place of Aib-3 yielded peptides 3a–c that preferred a β-sheet-like conformation. The impact of the ΔAA on peptide structure was independent of size, with small (ΔAla), medium-sized (Z-ΔAbu), and bulky (ΔVal) ΔAAs exerting similar effects. The proteolytic stabilities of 1 and its analogs were determined by incubation with Pronase. Z-ΔAbu and ΔVal increased the resistance of peptides to proteolysis when incorporated at the 3-position and had negligible impact on stability when placed at the 1-position, whereas ΔAla-containing peptides degraded rapidly regardless of position. Exposure of peptides 2a–c and 3a–c to the reactive thiol cysteamine revealed that ΔAla-containing peptides underwent conjugate addition at room temperature, while Z-ΔAbu- and ΔVal-containing peptides were inert even at elevated temperatures. These results suggest that both bulky and more accessible medium-sized ΔAAs should be valuable tools for bestowing rigidity and proteolytic stability on bioactive peptides.
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