Differential scanning calorimetric and spectroscopic studies on the unfolding of Momordica charantia lectin. Similar modes of thermal and chemical denaturation.
Thermal stability of Momordica charantia seed lectin (MCL) was investigated as a function of protein concentration, pH, scan rate, and at different ligand concentrations by using high-sensitivity differential scanning calorimetry (DSC). The DSC endotherm obtained at pH 7.4 consists of two entities with transition temperatures at ca. 333.7 K, and 338 K. The unfolding process is irreversible and could be described by a three-state model. For MCL tetramer DeltaH(c)/DeltaH(v) ratio is close to 4 for the first transition and approximately 2 for the second transition, suggesting that four and two cooperative units are involved in the first and second transitions, respectively. In the presence of lactose both transitions shifted to higher temperatures, suggesting that ligand binds preferentially to the native conformation of MCL. Endotherms recorded as a function of pH indicate that MCL is more stable at lower pH. Chemical unfolding of MCL, induced by Gdn.HCl, was investigated by monitoring the intrinsic fluorescence properties of the protein. The results obtained indicate that chemical denaturation of MCL can also be described by a three-state process, involving an intermediate populated at approximately 3-4 M Gdn.HCl. These observations suggest that the chemical and thermal unfolding processes are similar in that both of them proceed via an intermediate. The far UV and near UV CD spectra of MCL were nearly identical at different pH values and indicate that its secondary and tertiary structure do not change significantly with pH, suggesting that the structure of the protein is stable over a wide pH range.
Kavitha
M
M
University of Hyderabad, Andhra Pradesh, India.
Bobbili
Kishore Babu
KB
Swamy
Musti J
MJ
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D029777:Momordica charantia; D037121:Plant Lectins / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D008024:Ligands; D037121:Plant Lectins; D019791:Guanidine