Reducing Lambda Repressor to the Core

Lambda repressor fragment λ6−85* is one of thefastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ6−85* would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λblue1, is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ6−85* pseudo wild type and a well-defined computed structure.λ阻遏蛋白片段λ6−85*是目前已知折叠速率最快的小型蛋白片段之一。我们推测，移除λ6−85*的5个螺旋中的3个，可将该蛋白进一步简化至其最小折叠核心结构。分子动力学模拟筛选出两种仅由螺旋1与螺旋4通过短甘氨酸/丝氨酸连接肽连接的能量稳定简化结构，以及一种稳定性较弱的对照结构。我们通过圆二色性（circular dichroism）、荧光光谱法（fluorescence spectroscopy）与温度跳跃弛豫光谱法（temperature jump relaxation spectroscopy）对这三种多肽及其片段开展了实验研究，以解析其热力学与动力学特性。基于热变性实验结果，多肽稳定性的排序与理论预测结果相符。其中稳定性最优的双螺旋束蛋白λblue1是一种协同折叠的微型蛋白，其解链温度与折叠速率均与全长λ6−85*伪野生型一致，且拥有结构明确的计算预测模型。