Stepwise unfolding of Ribonuclease A by a biosurfactant.

The interaction of Ribonuclease A (RNase A) with the bile salt, sodium deoxycholate (NaDC) is meticulously investigated using various spectroscopic techniques. The binding isotherm constructed from the modulation of intrinsic tyrosine fluorescence of the protein following interaction with NaDC conspicuously reveals an intrinsically complex stepwise interaction process which proceeds through the formation of distinct conformational states of the protein. The conformational transitions are found to occur at c=2.2mM, and c=7.2mM of NaDC. These results are subsequently corroborated from the studies of excited-state relaxation dynamics of the intrinsic tyrosine residues of RNase A, and the modulations in fluorescence behavior of an extrinsic probe (ANS) bound to the protein. The far-UV circular dichroism spectral analyses unveil only nominal influence on the secondary structural element of the protein up to [NaDC]=c=2.2mM, which is then followed by marked disruption of the secondary structure of RNase A following further addition of NaDC. This clearly accounts for differential interaction behaviors of RNase A with the monomeric and micellar forms of NaDC (CMC of NaDC in aqueous buffer is estimated to be ∼3.0mM). In Region-I (up to c=2.2mM), the protein:surfactant interaction is argued to be predominantly governed by electrostatic/ionic interaction force. Subsequently, in Region-II (up to c=7.2mM) the RNase A:NaDC interaction accompanies major denaturation of the protein (∼17% loss of the secondary structure of RNase A at c=7.2mM) resulting in significant exposure of hydrophobic surfaces of the protein. However, the tertiary structure of the protein remains essentially unperturbed within the concentration regime of NaDC under study.