Abstract
Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C4mim]Cl and [C2OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference – nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.
Highlights
Molybdenum is a transition metal that is incorporated as a biologically active cofactor in a class of widely distributed proteins collectively known as molybdoenzymes [1]
The molecular chaperone PaoD is essential for the maturation of the periplasmic aldehyde oxidase PaoABC [12]
PaoD has been purified as a dimeric protein and could be successfully stabilized and concentrated in the presence of specific ionic liquids (IL) ([C4mim]Cl and [C2OHmim]PF6)
Summary
Molybdenum is a transition metal that is incorporated as a biologically active cofactor (molybdenum cofactor, Moco) in a class of widely distributed proteins collectively known as molybdoenzymes [1]. The crystal structures of several molybdoenzymes revealed that Moco is deeply buried inside the proteins, at the end of a funnelshaped passage giving access to the substrate [6]. This implied the requirement of specific chaperones for each molybdoenzyme, to facilitate the insertion of Moco [7]
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