Oligomerization, Conformational Stability and Thermal Unfolding of Harpin, HrpZ_Pss and Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-HrpZ_Pss

<div><p>HrpZ—a harpin from <i>Pseudomonas syringae</i>—is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZ_Pss from <i>Pseudomonas syringae</i> pv. <i>syringae</i> and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZ_Pss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZ_Pss and HrpZ_Pss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (∼10.0 kcal/mol) of HrpZ_Pss and C-214-HrpZ_Pss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities.</p>