Abstract
PDZ domains are one of the most important protein-protein interaction domains in human. While presenting a conserved three dimensional structure, a substantial number of PDZ domains display structural extensions suggested to be involved in their folding and binding mechanisms. The C-terminal α-helix extension (α3) of the third PDZ domain from PSD-95 (PDZ3) has been reported to have a role in function of the domain as well as in the stabilization of the native fold. Here we report an evaluation of the effect of the truncation of this additional helix on the folding and unfolding kinetics of PDZ3. Fluorescent variants of full length and truncated PDZ3 were produced and stopped-flow fluorescence measurements were made under different experimental conditions (pH, ionic strength and temperature) to investigate the folding kinetics of the respective variant. The results show that folding of PDZ3 is robust and that the mechanism is only marginally affected by the truncation, which contributes to a destabilization of the native state, but otherwise do not change the overall observed kinetics. Furthermore, the increase in the unfolding rate constants, but not the folding rate constant upon deletion of α3 suggests that the α-helical extension is largely unstructured in the folding transition state.
Highlights
PDZ domains are the most abundant protein interaction modules in human, being characterized by over 200 different domains[1,2,3,4,5]
Such a behavior was mainly assigned to the dynamic properties of the side chain of the domain, and led the authors to propose α3 to represent an element of allosteric regulation for PDZ3
In order to test the effect of the α-helical extension in the folding of PDZ3, in analogy to our previous work on this protein family[23,24,25,26,27,28], we used a fluorescent pseudo-wild type variant of PDZ3, where a Phe residue is replaced with a Trp in position 337 (F337W)
Summary
PDZ domains are the most abundant protein interaction modules in human, being characterized by over 200 different domains[1,2,3,4,5]. A comparison by NMR between the full-length and truncated constructs, where this contiguous structural element was deleted, suggested α3 to be responsible for the modulation of dynamic and binding properties of the PDZ domain[17,19,20]. Such a behavior was mainly assigned to the dynamic properties of the side chain of the domain, and led the authors to propose α3 to represent an element of allosteric regulation for PDZ3. The overall kinetic folding mechanism appears similar when the protein is challenged with acidic conditions, different ionic strengths and different temperatures
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