Acyl carrier proteins (ACPs) are central hubs in polyketide and fatty acid biosynthetic pathways, but the fast motions of the ACP’s phosphopantetheine (Ppant) arm make its conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we report that converting the terminal thiol of E. coli ACP’s Ppant arm into a thiocyanate activates this site to form a selective crosslink with the active site cysteine of its partner ketoacyl synthase (KS; FabF). The reaction releases a cyanide anion, which can be detected by infrared spectroscopy. This represents a practical and generalizable method to obtain and visualize ACP-protein complexes relevant to biocatalysis and will be valuable in future structural and engineering studies.

Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogelforming peptide, SgI 37-49 . Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI 37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI 37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.

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allows Lif to bind LipA despite the fickleness of structure. Structural details of the process are studied by the fluorescence spectroscopic toolkit: a FRET distance landscape is investigated by ensemble lifetime-resolved FRET (eTCSPC), the timescales of dynamics by correlation techniques (filtered FCS) and overall FRET populations and dynamics by single-molecule MFD studies. The combination of these techniques allows drawing a kinetic scheme within the limiting states of the conformational ensemble of Lif, unraveling the mechanism of Lif action. The study is supported by all-atom MD simulations. The combination of accurate FRET, MFD, FRET positioning and screening (FPS) and fFCS with MD simulations in a hybrid approach provides in-depth insight in the complexity of the conformational dynamics of flexible proteins and protein complexes.