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PCR Cloning and Subcloning> | … Cloning and purification of wild type PimA and PimA mutants PimAR144C and PimAT126C-V359C-R144C mutants of PimA from Mycobacterium smegmatis, were obtained from GenScript using pET29a-pimA as the DNA template (26) … | Get A Quote |
Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix-to-β-strand and β-strand-to-α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of millisec... More