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

HrpZ-a harpin from Pseudomonas syringae-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 HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss 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 HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities

Involvement of mitochondria and metacaspase elevation in harpin_Pss-induced cell death of Saccharomyces cerevisiae

Expression of a proteinaceous elicitor harpinPss, encoded by hrpZ of Pseudomonas syringae pv. syringae 61, under GAL1 promoter in Saccharomyces cerevisiae Y187 resulted in galactose-inducible yeast cell death (YCD). Extracellular treatment of harpin did not affect the growth of yeast. The observed YCD was independent of the stage of cell cycle. “Petite” mutant of S. cerevisiae Y187 pYEUT-hrpZ was insensitive to cell death indicating the involvement of mitochondria in this YCD. Loss in mitochondrial potential, but no leakage of Cytochrome c from mitochondria into the cytosol, were notable features in harpinPss-induced YCD. Cyclosporin A had no effect on hrpZ expressing yeast cells, further confirmed that there was no release of Cytochrome c. Elevation of caspase activity has been reported for the first time in this form of cell death induced by harpin expression. Release of reactive oxygen species and clear loss of membrane integrity were evident with the absence of nuclear fragmentation and chromosomal condensation, while annexin V and propidium iodide staining showed features typical of necrosis

Thermally stable harpin, HrpZ_Pss 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 λ_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^-(~10%) and Cs+ (negligible) was considerably lower. In the presence of 6.0 M guanidine hydrochloride (GdnHCl) the emission λ_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

Biophysical investigations on the aggregation and thermal unfolding of harpin<SUB>Pss</SUB> 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 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 &#945;-helical, remains unchanged until the onset of transition 2, above which the &#945;-helical content decreases while the &#946;-sheet content increases. Dynamic light scattering measurements yielded the hydrodynamic radius (Rh) of harpinPss as room temperature as 20.54 &#177; 6.19 nm, which decreases to 9.35 nm at 61 &#176;C. These results could be explained in terms of the following thermal unfolding pathway for harpinPss: oligomer &#8594; dimer &#8594; partially unfolded dimer &#8594; 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

Differential scanning calorimetry.

<p>DSC thermograms of C-214-HrpZ_Pss (A) and full length HrpZ_Pss (B). The scan rate was 60^o.h⁻¹. The change in heat capacity (ΔC_p) of the native protein and the denatured state are shown. Deconvolution of DSC thermogram of C-214-HrpZ_Pss (C) and full length HrpZ_Pss (D). The experimentally obtained thermograms are shown as solid lines, individual transitions deduced from deconvolution analysis are shown as dashed lines and the sum of the transitions obtained from deconvolution analysis are shown as dotted lines.</p

Thermodynamic parameters for the thermal unfolding of C-214-HrpZ_Pss.

<p>Values given are averages obtained from 3 independent measurements. Values in parentheses correspond to the thermal unfolding of full length HrpZ_Pss<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109871#pone.0109871-Tarafdar1" target="_blank">[15]</a>. *C-214-HrpZ_Pss does not have transition 3.</p><p>Thermodynamic parameters for the thermal unfolding of C-214-HrpZ_Pss.</p

Differential oligomerization of C-214-HrpZ_Pss and full length HrpZ_Pss studied by AFM.

<p>C-214-HrpZ_Pss forms non specific aggregates (<b>A</b>), whereas full length HrpZ_Pss is characterized by the formation of both non-specific and fibrillar aggregates (<b>B</b>). Scale bar: 0.5 µm.</p

Circular dichroism spectroscopy.

<p>A) Far-UV CD spectra of C-214-HrpZ_Pss (solid line) and full length HrpZ_Pss (dashed line) at 25°C. The dotted line corresponds to the calculated fit obtained by using the CDSSTR program for C-214-HrpZ_Pss. B) Temperature dependence of α-helical content in C-214-HrpZ_Pss.</p

Results of CD spectral analysis of C-214-HrpZ_Pss.

<p>The secondary structural components of the full-length HrpZ_Pss<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109871#pone.0109871-Tarafdar1" target="_blank">[15]</a> are given in parentheses.</p><p>Results of CD spectral analysis of C-214-HrpZ_Pss.</p

Tryptophan exposure and thermal unfolding.

<p>(A) Stern-Volmer plots for the quenching of the intrinsic fluorescence of C-214-HrpZ_Pss with acrylamide (□), iodide ion (○) and cesium ion (▴). Temperature dependence of (B) tryptophan emission maximum, and (C) fluorescence intensity at emission maximum, of C-214-HrpZ_Pss.</p