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 (Gn peptides) have a higher loop closure propensity compared to DA. Comparison of monomeric and dimeric Rop simulations suggests that dimeric Rop necessarily requires αL conformation to be sampled at D30/G30 position in the turn region. Since glycine (at position 30) can readily adopt αL conformation, Gn 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.

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► Rop is a dimeric four-helix bundle with helix-turn-helix motif.
► Its folding rates are significantly enhanced by insertion of glycine residues in the turn region.
► MD simulations of glycine inserted turn mutant peptides show higher loop closure propensities.
► Simulations show that it is mandatory for the position 30 in the turn region of the dimeric Rop to adopt αL conformation.
► Loop propensity plays an important role in determining the overall folding rates of Rop.