Kinetic stability plays a dominant role in the denaturant-induced unfolding of Erythrina indica lectin.
The urea-induced denaturation of dimeric Erythrina indica lectin (EIL) has been studied at pH 7.2 under equilibrium and kinetic conditions in the temperature range of 40-55 degrees C. The structure of EIL is largely unaffected in this temperature range in absence of denaturant, and also in 8 M urea after incubation for 24 h at ambient temperature. The equilibrium denaturation of EIL exhibits a monophasic unfolding transition from the native dimer to the unfolded monomer as monitored by fluorescence, far-UV CD, and size-exclusion FPLC. The thermodynamic parameters determined for the two-state unfolding equilibrium show that the free energy of unfolding (DeltaGu, aq) remains practically same between 40 and 55 degrees C, with a value of 11.8 +/- 0.6 kcal mol(-1) (monomer units). The unfolding kinetics of EIL describes a single exponential decay pattern, and the apparent rate constants determined at different temperatures indicate that the rate of the unfolding reaction increases several fold with increase in temperature. The presence of probe like external metal ions (Mn2+, Ca2+) does not influence the unfolding reaction thermodynamically or kinetically; however, the presence of EDTA affects only kinetics. The present results suggest that the ability of EIL to preserve the structural integrity against the highly denaturing conditions is linked primarily to its kinetic stability, and the synergic action of heat and denaturant is involved in the unfolding of the protein.
Ghosh
Sujit
S
Department of Chemistry, Presidency College, 86/1 College Street, Kolkata 700 073, India.
Mandal
Dipak K
DK
D002118:Calcium / Q000737:chemistry; D002942:Circular Dichroism; D019281:Dimerization; D004895:Erythrina / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D037121:Plant Lectins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
D037121:Plant Lectins; C045629:erythrina lectin; D008274:Magnesium; D002118:Calcium