Biophysical investigations on the aggregation and thermal unfolding of harpin < inf > Pss < /inf > and identification of leucine-zipper-like motifs in harpins

Abstract

Harpins are heat-stable, glycine-rich proteins secreted by Gram-negative bacteria, which induce hypersensitive response (HR) in non-host plants. In this study, the thermal unfolding and aggregation of harpinPss from Pseudomonas syringae pv. syringae have been investigated by biophysical approaches. Differential scanning calorimetric studies indicate that thermal unfolding of harpinPss is a complex process involving three distinct transitions. CD spectroscopy revealed that the secondary structure of the protein, which is predominantly α-helical, remains unchanged until the onset of transition 2, above which the α-helical content decreases while the β-sheet content increases. Dynamic light scattering measurements yielded the hydrodynamic radius (Rh) of harpinPss as room temperature as 20.54 ± 6.19 nm, which decreases to 9.35 nm at 61 °C. These results could be explained in terms of the following thermal unfolding pathway for harpinPss: oligomer→dimer→partially unfolded dimer→unfolded monomer. Sequence analysis indicated the presence of at least two leucine-zipper-like motifs in harpinPss and several other harpins, whereas computational modelling studies suggest that most of them are located on helices present on protein surfaces, suggesting that they can take part in the formation of oligomeric aggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. © 2009 Elsevier B.V. All rights reserved.