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Oligomerization, conformational stability and thermal unfolding of Harpin, HrpZPss and its hypersensitive response-inducing c-terminal fragment, C-214-HrpZPss.

Tarafdar PK, Vedantam LV, Sankhala RS, Purushotham P, Podile AR, Swamy MJ - PLoS ONE (2014)

Bottom Line: Both C-214-HrpZPss and HrpZPss were found to form oligomers.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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Hyderabad, Hyderabad, India.

ABSTRACT
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.

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Conformational stability curves of C-214HrpZPss and HrpZPss.Solid lines correspond to dimer - unfolded state transition (ΔGDi-U). Values of ΔG2 and ΔG3 for HrpZPss corresponding to transition 2 and 3 were calculated using equations 1 and 2 (see text for more details).
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pone-0109871-g006: Conformational stability curves of C-214HrpZPss and HrpZPss.Solid lines correspond to dimer - unfolded state transition (ΔGDi-U). Values of ΔG2 and ΔG3 for HrpZPss corresponding to transition 2 and 3 were calculated using equations 1 and 2 (see text for more details).

Mentions: The conformation stability curve (i.e. the dependence of ΔG of unfolding with temperature) describes the thermodynamic behaviour of the equilibrium of unfolding. The stability curve of a protein can be drawn with confidence using the modified Gibbs-Helmholtz equation. For N↔U equilibrium, where N is the native state and U is the unfolded state, the equation is: where Tm is the melting temperature and ΔHc is the enthalpy of unfolding at this temperature. ΔCp is the heat capacity change, which is independent of solution conditions and temperature between 20 and 80°C [27], [28]. The equation will change for N2↔2U equilibrium [29], [30]: where Pt is protein monomer concentration. Fig 6A and 6B display free energy profiles of C-214-HrpZPss and HrpZPss. The thermal unfolding profile of HrpZPss is complex and does not follow a simple two-state transition. Results of studies employing various biophysical approaches suggested the following pathway of unfolding: Oligomer → dimer → partially unfolded dimer → unfolded monomer [15]. Using the Gibbs-Helmholtz equation (1), ΔG for the dimer to partially unfolded dimer (ΔG2) at 25°C was estimated to be 1.04 kcal/mol (Fig. 6B). In obtaining the ΔG2 we assume that there is no significant change in heat capacity (Cp) between transition 1 and transition 2. The first transition was attributed to the dissociation of oligomer with different oligomerization states to dimer [15]. HrpZPss exists as polydisperse oligomers and the oligomer nucleates from dimer [14]. The secondary structure of the protein at 25°C (before the onset of transition 1) and at 45°C (before the onset of transition 2) is similar and the fluorescence emission maximum of the protein at both the temperatures was identical [22], suggesting that the Cp approximation is valid. The ΔG3 of HrpZPss was determined using equation 2 and plotted as dash-dotted line (Fig. 6B). Since we could not determine Cp of unfolded monomer accurately, we keep ΔCp as zero. It is a reasonable approximation as the tryptophan environment of the partially unfolded dimer is very close to that of the unfolded monomer [22]. ΔGDi-U of HrpZPss was plotted as solid line by combining ΔG2 and ΔG3. The ΔGDi-U for C-214-HrpZPss was determined using equation 2. For both proteins, the ΔGDi-U at 25°C is comparable; 9.9 kcal/mol for HrpZPss and 10.7 kcal/mol for C-214-HrpZPss.


Oligomerization, conformational stability and thermal unfolding of Harpin, HrpZPss and its hypersensitive response-inducing c-terminal fragment, C-214-HrpZPss.

Tarafdar PK, Vedantam LV, Sankhala RS, Purushotham P, Podile AR, Swamy MJ - PLoS ONE (2014)

Conformational stability curves of C-214HrpZPss and HrpZPss.Solid lines correspond to dimer - unfolded state transition (ΔGDi-U). Values of ΔG2 and ΔG3 for HrpZPss corresponding to transition 2 and 3 were calculated using equations 1 and 2 (see text for more details).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264689&req=5

pone-0109871-g006: Conformational stability curves of C-214HrpZPss and HrpZPss.Solid lines correspond to dimer - unfolded state transition (ΔGDi-U). Values of ΔG2 and ΔG3 for HrpZPss corresponding to transition 2 and 3 were calculated using equations 1 and 2 (see text for more details).
Mentions: The conformation stability curve (i.e. the dependence of ΔG of unfolding with temperature) describes the thermodynamic behaviour of the equilibrium of unfolding. The stability curve of a protein can be drawn with confidence using the modified Gibbs-Helmholtz equation. For N↔U equilibrium, where N is the native state and U is the unfolded state, the equation is: where Tm is the melting temperature and ΔHc is the enthalpy of unfolding at this temperature. ΔCp is the heat capacity change, which is independent of solution conditions and temperature between 20 and 80°C [27], [28]. The equation will change for N2↔2U equilibrium [29], [30]: where Pt is protein monomer concentration. Fig 6A and 6B display free energy profiles of C-214-HrpZPss and HrpZPss. The thermal unfolding profile of HrpZPss is complex and does not follow a simple two-state transition. Results of studies employing various biophysical approaches suggested the following pathway of unfolding: Oligomer → dimer → partially unfolded dimer → unfolded monomer [15]. Using the Gibbs-Helmholtz equation (1), ΔG for the dimer to partially unfolded dimer (ΔG2) at 25°C was estimated to be 1.04 kcal/mol (Fig. 6B). In obtaining the ΔG2 we assume that there is no significant change in heat capacity (Cp) between transition 1 and transition 2. The first transition was attributed to the dissociation of oligomer with different oligomerization states to dimer [15]. HrpZPss exists as polydisperse oligomers and the oligomer nucleates from dimer [14]. The secondary structure of the protein at 25°C (before the onset of transition 1) and at 45°C (before the onset of transition 2) is similar and the fluorescence emission maximum of the protein at both the temperatures was identical [22], suggesting that the Cp approximation is valid. The ΔG3 of HrpZPss was determined using equation 2 and plotted as dash-dotted line (Fig. 6B). Since we could not determine Cp of unfolded monomer accurately, we keep ΔCp as zero. It is a reasonable approximation as the tryptophan environment of the partially unfolded dimer is very close to that of the unfolded monomer [22]. ΔGDi-U of HrpZPss was plotted as solid line by combining ΔG2 and ΔG3. The ΔGDi-U for C-214-HrpZPss was determined using equation 2. For both proteins, the ΔGDi-U at 25°C is comparable; 9.9 kcal/mol for HrpZPss and 10.7 kcal/mol for C-214-HrpZPss.

Bottom Line: Both C-214-HrpZPss and HrpZPss were found to form oligomers.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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Hyderabad, Hyderabad, India.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus