PDF(Pubmed)
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 Alanine31 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.
摘要：
突变对蛋白质结构的影响通常是局部化的和轻微的。发现可以单手改变蛋白质结构的折叠和/或拓扑结构的突变是罕见的例外。引物同源二聚体阻遏物（Rop）蛋白的A31P突变体就是这样一个例外：这种单突变-并通过两个独立的晶体结构测定证明-可以将规范（左手/全反平行）4-α-Rop的螺旋束转换为新形式（右手/混合平行和反平行束），显示出以前未观察到的“平分U拓扑”。理解这种突变对Rop折叠的戏剧性影响的主要问题是理解它的存在：大多数计算方法似乎都同意突变应该没有明显的影响，大多数能量最小化方法和蛋白质结构预测方案表明该突变与天然Rop结构完全一致，只需要在突变位点进行局部和微小的改变。在这里，我们使用两个长（每个10μs）分子动力学模拟来比较天然Rop的稳定性和动力学与与天然Rop相同但携带此单个Alanine31到脯氨酸突变的假设结构。对这两种轨迹的比较分析令人信服地表明，与能量最小化的迹象相反，但与实验数据一致，这种假设的原生A31P结构是不稳定的，随着它的转向区域几乎完全展开，即使在我们用于本研究的相对温和的320KNpT模拟下。我们讨论了这些发现对A31P突变体折叠的意义，特别是关于提出的双漏斗能源景观模型。
