Tryptophan Fluorescence as a Probe for Conformational Dynamics in Zinc Solute Binding Proteins

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Bacterial ATP binding cassette (ABC) transporters are important for the uptake of various nutrients, including essential metals such as zinc. These systems require an extracellular solute binding protein (SBP) to bind the substrate with high affinity and specificity and deliver it to the membrane permease for transport. Generally, substrate binding elicits a change from an open to closed conformation that is likely important for permease recognition and transport. However, crystal structures for the metal‐bound and metal‐free forms of several zinc SBPs show relatively subtle or even negligible conformational changes that accompany metal binding. Thus, solution studies are needed for a thorough understanding of the role of conformational dynamics in substrate binding and transport. Here we investigate conformational states and dynamics in a group of zinc SBPs using tryptophan fluorescence intensity and lifetime as a sensitive reporter for the environment of this residue. Useful changes were observed in homologues of the protein AztC from Paracoccus denitrificans and the human pathogen Citrobacter koseri, whereas fluorescence from Citrobacter koseri ZnuA showed no change in fluorescence behavior upon zinc binding. These data combined with isothermal titration calorimetry (ITC) suggest a dynamic equilibrium of at least two conformational states in the apo form and compensatory changes in the holo form that provide for a significant entropic contribution to zinc binding. Correlation with available crystal structures suggests that the formation of a Trp‐Phe pi‐stacking interaction in AztC that is absent in ZnuA may mediate the observed changes in fluorescence. The conformational dynamics identified here highlight diversity in binding mechanisms for zinc SBPs with relevance to the exploitation of these proteins as potential antibiotic drug targets.