The role of loop closure propensity in the refolding of Rop protein probed by molecular dynamics simulations.

Rop protein is a homo-dimer of helix-turn-helix and has relatively slow folding and unfolding rates compared to other dimeric proteins of similar size. Fluorescence studies cited in literature suggest that mutation of turn residues D30-A31 to G30-G31 (Gly₂) increases its folding and unfolding rates considerably. A further increase in number of glycines in the turn region results in decrease of folding rates compared to Gly₂ mutant. To understand the effect of glycine mutation on folding/unfolding rates of Rop and the conformational nature of turn region involved in formation of early folding species, we performed molecular dynamics simulations of turn peptides, ²⁵KLNELDADEQ³⁴ (DA peptide), ²⁵KLNELGGDEQ³⁴ (G₂ peptide), ²⁵KLNELGGGDEQ³⁵ (G₃ peptide) and ²⁵KLNELGGGEQ³⁴ (G₃(') peptide) from Rop at 300 K. Further Wt-Rop and mutant G₂-Rop monomers and dimers were also studied separately by molecular dynamics simulations. Our results show that glycine based peptides (G(n) peptides) have a higher loop closure propensity compared to DA. Comparison of monomeric and dimeric Rop simulations suggests that dimeric Rop necessarily requires α(L) conformation to be sampled at D30/G30 position in the turn region. Since glycine (at position 30) can readily adopt α(L) conformation, G(n) loop plays a dual role in both facilitating loop closure as well as facilitating reorganization/packing of helices required for structural adjustment during dimer formation in the folding of Rop. Based on our simulation results and available literature, we suggest a tentative kinetic model for Rop folding which allows us to estimate the contribution of loop closure propensity to the overall folding rates.