The plastoquinone (Q(B)) binding niche of the Photosystem II (PSII) D1 protein is the subject of intense research due to its capability to bind also anthropogenic pollutants. In this work, the Chlamydomonas reinhardtii D1 primary structure was used as a template to computationally design novel peptides enabling the binding of the herbicide atrazine. Three biomimetic molecules, containing the Q(B)-binding site in a loop shaped by two alpha-helices, were reconstituted by automated protein synthesis, and their structural and functional features deeply analysed by biophysical techniques. Standing out among the others, the biomimetic mutant peptide, D1pepMut, showed high ability to mimic the D1 protein in binding both Q(B) and atrazine. Circular dichroism spectra suggested a typical properly-folded alpha-helical structure, while isothermal titration calorimetry (ITC) provided a complete thermodynamic characterization of the molecular interaction. Atrazine binds to the D1pepMut with a high affinity (K-d = 2.84 mu M), and a favourable enthalpic contribution (Delta H = -11.9 kcal mol(-1)) driving the interaction. Fluorescence spectroscopy assays, in parallel to ITC data, provided hyperbolic titration curves indicating the occurrence of a single atrazine binding site. The binding resulted in structural stabilisation of the D1pepMut molecule, as suggested by atrazine-induced cooperative profiles for the fold-unfold transition. The interaction dynamics and the structural stability of the peptides in response to the ligand were particularly considered as mandatory parameters for biosensor/biochip development. These studies paved the way to the set-up of an array of synthetic mutant peptides with a wide range of affinity towards different classes of target analytes, for the development of optical nanosensing platforms for herbicide detection.
|Titolo:||Design and biophysical characterization of atrazine-sensing peptides mimicking the Chlamydomonas reinhardtii plastoquinone binding niche|
|Autori interni:||POLTICELLI, Fabio|
|Data di pubblicazione:||2013|
|Rivista:||PHYSICAL CHEMISTRY CHEMICAL PHYSICS|
|Appare nelle tipologie:||1.1 Articolo in rivista|