Abstract
The effect of mutations on protein structures is usually rather localized and minor. Finding a mutation that can single-handedly change the fold and/or topology of a protein structure is a rare exception. The A31P mutant of the homodimeric Repressor of primer (Rop) protein is one such exception: This single mutation ─and as demonstrated by two independent crystal structure determinations─ can convert the canonical (left-handed/all-antiparallel) 4-α-helical bundle of Rop to a new form (right-handed/mixed parallel and antiparallel bundle) displaying a previously unobserved "bisecting U" topology. The main problem with understanding the dramatic effect of this mutation on the folding of Rop is to understand its very existence: Most computational methods appear to agree that the mutation should have had no appreciable effect, with the majority of energy minimization methods and protein structure prediction protocols indicating that this mutation is fully consistent with the native Rop structure, requiring only a local and minor change at the mutation site. Here we use two long (10 μs each) molecular dynamics simulations to compare the stability and dynamics of the native Rop versus a hypothetical structure that is identical with the native Rop but is carrying this single Alanine(31) to Proline mutation. Comparative analysis of the two trajectories convincingly shows that, in contrast to the indications from energy minimization ─but in agreement with the experimental data─, this hypothetical native-like A31P structure is unstable, with its turn regions almost completely unfolding, even under the relatively mild 320 K NpT simulations that we have used for this study. We discuss the implication of these findings for the folding of the A31P mutant, especially with respect to the proposed model of a double-funneled energy landscape.