Thermally stable harpin, HrpZPss 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 HrpZPss, secreted by Pseudomonas syringae pv. syringae, have been characterized by fluorescence spectroscopic studies. Emission λmax 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 λmax 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 HrpZPss 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.
School of Chemistry, University of Hyderabad, Hyderabad 500046, India.
Pradip K
PK
Tarafdar
Lakshmi Vasudev
LV
Vedantam
Appa Rao
AR
Podile
Musti J
MJ
Swamy
D020106:Acrylamide / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000187:drug effects; D002152:Calorimetry, Differential Scanning; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D011489:Protein Denaturation; D055550:Protein Stability; D058767:Protein Unfolding / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; C087295:HrpZ protein, Pseudomonas syringae; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea; D019791:Guanidine
Chemical unfolding; Fluorescence quenching; Harpin(Pss); Linear extrapolation model; Thermal unfolding