Abstract
A growing body of literature on intrinsically disordered proteins (IDPs) led scientists to rethink the structure-function paradigm of protein folding. Enzymes are often considered an exception to the rule of intrinsic disorder (ID), believed to require a unique structure for catalysis. However, recent studies revealed the presence of disorder in several functional native enzymes. In the present work, we address the importance of dynamics for catalysis, by investigating the relationship between folding and activity in Sporosarcina pasteurii UreG (SpUreG), a P-loop GTPase and the first discovered native ID enzyme, involved in the maturation of the nickel-containing urease. The effect of denaturants and osmolytes on protein structure and activity was analyzed using circular dichroism (CD), Site-Directed Spin Labeling (SDSL) coupled to EPR spectroscopy, and enzymatic assays. Our data show that SpUreG needs a “flexibility window” to be catalytically competent, with both too low and too high mobility being detrimental for its activity.
Highlights
The lack of tertiary structure in functional proteins is an exciting discovery of the last couple of decades[1, 2]
We address the relationship between the structural plasticity and the enzymatic activity in Sporosarcina pasteurii UreG (SpUreG), by perturbing the protein conformational landscape using denaturants and osmolytes and investigating its structure and function with circular dichroism (CD), Site-Directed Spin Labeling (SDSL) coupled to Electron Paramagnetic Resonance (EPR) spectroscopy, and enzymatic assays
The shape of the continuous wave EPR spectrum reflects the mobility of the nitroxide, which acts as a sensitive reporter of the local environment and motion of the protein[26,27,28]
Summary
The lack of tertiary structure in functional proteins is an exciting discovery of the last couple of decades[1, 2]. On the other hand, are traditionally viewed as an exception to the rule, through the lens of the rigid “lock-and-key” model This idea, amended by the observation that enzymes may shift between diverse conformational states upon substrate binding and conversion[3], is based on the belief that, if some protein flexibility is necessary for enzymes upon catalysis, major disorder is unfavorable for catalytic efficiency[4]. UreG is a GTP hydrolase that assists nickel delivery into nickel-dependent urease, a pathogenic factor for several bacteria and fungi This GTPase functions in complex with three accessory proteins, namely UreF, UreD and UreE, that cooperate for urease activation forming a multimeric molecular chaperone[17].
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