Department of Chemistry, University of Utrecht, Utrecht, The Netherlands.
J Am Chem Soc. 2010 Mar 17;132(10):3452-60. doi: 10.1021/ja909347x.
Partitioning properties of transmembrane (TM) polypeptide segments directly determine membrane protein folding, stability, and function, and their understanding is vital for rational design of membrane active peptides. However, direct determination of water-to-bilayer transfer of TM peptides has proved difficult. Experimentally, sufficiently hydrophobic peptides tend to aggregate, while atomistic computer simulations at physiological temperatures cannot yet reach the long time scales required to capture partitioning. Elevating temperatures to accelerate the dynamics has been avoided, as this was thought to lead to rapid denaturing. However, we show here that model TM peptides (WALP) are exceptionally thermostable. Circular dichroism experiments reveal that the peptides remain inserted into the lipid bilayer and are fully helical, even at 90 degrees C. At these temperatures, sampling is approximately 50-500 times faster, sufficient to directly simulate spontaneous partitioning at atomic resolution. A folded insertion pathway is observed, consistent with three-stage partitioning theory. Elevated temperature simulation ensembles further allow the direct calculation of the insertion kinetics, which is found to be first-order for all systems. Insertion barriers are DeltaH(in)(double dagger) = 15 kcal/mol for a general hydrophobic peptide and approximately 23 kcal/mol for the tryptophan-flanked WALP peptides. The corresponding insertion times at room temperature range from 8.5 mus to 163 ms. High-temperature simulations of experimentally validated thermostable systems suggest a new avenue for systematic exploration of peptide partitioning properties.
跨膜（TM）多肽片段的分区性质直接决定膜蛋白的折叠、稳定性和功能，因此深入了解这些性质对于理性设计膜活性肽至关重要。然而，直接测定 TM 肽的水到双层的转移一直具有挑战性。实验上，足够疏水的肽往往会聚集，而在生理温度下进行原子级计算机模拟还不能达到捕捉分区所需的长时间尺度。升高温度以加速动力学过程的方法已经被避免了，因为这被认为会导致快速变性。然而，我们在这里表明，模型 TM 肽（WALP）具有异常的热稳定性。圆二色性实验表明，即使在 90°C 时，肽仍插入脂质双层并完全呈螺旋状。在这些温度下，采样速度大约快 50-500 倍，足以直接以原子分辨率模拟自发分区。观察到折叠的插入途径，与三阶段分区理论一致。升高温度的模拟组合进一步允许直接计算插入动力学，发现所有系统都是一级动力学。对于一般的疏水肽，插入势垒为 DeltaH(in)(double dagger) = 15 kcal/mol，而色氨酸侧翼的 WALP 肽约为 23 kcal/mol。在室温下的相应插入时间范围从 8.5 mus 到 163 ms。对实验验证的热稳定系统的高温模拟为系统探索肽分区性质提供了新途径。
