Thermally stable harpin, HrpZPss is sensitive to chemical denaturants: Probing tryptophan environment, chemical and thermal unfolding by fluorescence spectroscopy

Harpins - a group of proteins that elicit hypersensitive response (HR) in non-host plants - are secreted by certain Gram-negative plant pathogenic bacteria upon interaction with the plant. In the present study, the microenvironment and solvent accessibility of the sole tryptophan residue (Trp-167) in harpin HrpZ(Pss), secreted by Pseudomonas syringae pv. syringae, have been characterized by fluorescence spectroscopic studies. Emission lambda(max) of the native protein at 328 nm indicates that Trp-167 is buried in a hydrophobic region in the interior of the protein matrix. Significant quenching (53%) was seen with the neutral quencher, acrylamide at 0.5 M concentration, whereas quenching by ionic quenchers, I- (similar to 10%) and Cs+ (negligible) was considerably lower. In the presence of 6.0 M guanidine hydrochloride (GdnHCl) the emission lambda max shifted to 350.5 nm, and quenching by both neutral and ionic quenchers increased significantly, suggesting complete exposure of the indole side chain to the aqueous medium. Fluorescence studies on the thermal unfolding of HrpZ(Pss) are fully consistent with a complex thermal unfolding process and high thermal stability of this protein, inferred from previous differential scanning calorimetric and dynamic light scattering studies. However, the protein exhibits low resistance to chemical denaturants, with 50% unfolding seen in the presence of 1.77 M GdnHCl or 3.59 M urea. The ratio of m value, determined from linear extrapolation model, for GdnHCl and urea-induced unfolding was 1.8 and suggests the presence of hydrophobic interactions, which could possibly involve leucine zipper-like helical regions on the surface of the protein. (C) 2013 Elsevier Masson SAS. All rights reserved.