Biophysical investigations on the aggregation and thermal unfolding of harpin(Pss) and identification of leucine-zipper-like motifs in harpins.
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 harpin(Pss) from Pseudomonas syringae pv. syringae have been investigated by biophysical approaches. Differential scanning calorimetric studies indicate that thermal unfolding of harpin(Pss) is a complex process involving three distinct transitions. CD spectroscopy revealed that the secondary structure of the protein, which is predominantly alpha-helical, remains unchanged until the onset of transition 2, above which the alpha-helical content decreases while the beta-sheet content increases. Dynamic light scattering measurements yielded the hydrodynamic radius (R(h)) of harpin(Pss) as room temperature as 20.54 + or - 6.19 nm, which decreases to 9.35 nm at 61 degrees C. These results could be explained in terms of the following thermal unfolding pathway for harpin(Pss): oligomer-->dimer-->partially unfolded dimer-->unfolded monomer. Sequence analysis indicated the presence of at least two leucine-zipper-like motifs in harpin(Pss) 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.
Tarafdar
Pradip K
PK
School of Chemistry, University of Hyderabad, Hyderabad-500046, India.
Vedantam
Lakshmi Vasudev
LV
Kondreddy
Anil
A
Podile
Appa Rao
AR
Swamy
Musti J
MJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D055592:Biophysical Phenomena; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003224:Congo Red / Q000737:chemistry; D006358:Hot Temperature; D016350:Leucine Zippers / Q000235:genetics; D008027:Light; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D016415:Sequence Alignment
D001425:Bacterial Outer Membrane Proteins; C075462:harpin protein, Erwinia amylovora; D003224:Congo Red