Delineating the conformational dynamics of intermediate structures on the unfolding pathway of β-lactoglobulin in aqueous urea and dimethyl sulfoxide

The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of β-lactoglobulin (β-LgA) in aqueous mixture of 8 M urea and 8 M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of β-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of ∼3.0 kcal mol⁻¹. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around ∼30–35 ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (∼50–150 ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of ∼6.0 kcal mol⁻¹. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of β-LgA in urea and DMSO. Abbreviationsβ-LgAβ-lactoglobulinDMSOdimethyl sulfoxideFELfree-energy landscapeGdmClguanidinium chlorideIintermediate stateMGmolten globule statePMEparticle mesh EwaldQfraction of native contactsRMSDroot mean square deviationRMSFroot mean square fluctuation<i>R</i>_gradius of gyrationSASAsolvent Accessible Surface AreascSASAthe side chain SASATrptryptophan β-lactoglobulin dimethyl sulfoxide free-energy landscape guanidinium chloride intermediate state molten globule state particle mesh Ewald fraction of native contacts root mean square deviation root mean square fluctuation radius of gyration solvent Accessible Surface Area the side chain SASA tryptophan Communicated by Ramaswamy H. Sarma