Protein-surfactant interaction: sodium dodecyl sulfate-induced unfolding of ribonuclease A.
Protein-surfactant interaction is widely studied to understand stability and structural changes in proteins. In this Article, we have investigated SDS-induced unfolding of RNase A using absorbance, intrinsic fluorescence of the protein, anisotropy, TNS fluorescence, and near- and far-UV circular dichroism. Unfolding titration curves obtained from the absorbance and fluorescence changes were fitted into a five-state protein unfolding model by assuming formation of three intermediate states. Free energy changes and m-values of all four transitions between the native and unfolded state were evaluated. The transitions are categorized into two different regions. Region I, up to 0.5 mM of SDS, involves ionic interaction between the protein and SDS where the secondary and tertiary structure of the protein is altered to a less extent. In region II, hydrophobic interaction dominates and has two distinct transitions. The first transition arises from the aggregation of surfactant molecules around the protein hydrophobic sites. In the following transition, the micelles probably expand more, and a few more hydrophobic sites are occupied by the surfactant. In this region, the tertiary contacts are completely broken, and almost 50% of the secondary structure is lost. The aggregation of SDS around the protein starts well below the CMC. These conformational changes can be explained by the necklace and beads model, and the free energy of formation of such a complex for the RNase A-SDS system is found to be 5.2 (±1.0) kcal mol(-1). The probable interaction sites and the mechanism of unfolding have been discussed in detail.
Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, India.
K Tejaswi
KT
Naidu
N Prakash
NP
Prabhu
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D057927:Hydrophobic and Hydrophilic Interactions; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000737:chemistry
D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D012259:Ribonuclease, Pancreatic