Exploring the energy landscape of GFP by single-molecule mechanical experiments
Exploring the energy landscape of GFP by single-molecule mechanical experiments
复制标题
DOI：
10.1073/pnas.0404549101
复制
发表时间：
2004-11-16
影响因子：
11.1
通讯作者：
Rief, M
中科院分区：
文献类型：
--
作者：
Dietz, H;Rief, M
我们使用单分子力光谱来驱动单个GFP分子从原始状态通过其复杂的能量景观进入完全展开状态。与许多较小的蛋白质不同，GFP的机械展开是通过随后的两种中间状态进行的。从原生态到第一中间态的转变发生在大约35 pN的热平衡附近，其特征是7个残基的n端α -螺旋从β桶脱离。我们测量与这一转变相关的平衡自由能成本为22 k(B)T。这个小a-螺旋的分离完全破坏了GFP的热力学稳定性，即使β -管仍然完好无损，可以承受载荷。然而，在毫秒时间尺度上，蛋白质的机械稳定性是由打开β -桶脱离这种热力学不稳定的中间状态的激活屏障决定的。高带宽、时间分辨的测量表明，在β -桶展开时，悬臂弛豫相位显示出第二个亚稳态的机械中间体，其中一条完整的β -链从桶中分离出来。力分布和寿命的定量分析导致通过粗略的能量景观复杂的机械展开路径的详细图片。
We use single-molecule force spectroscopy to drive single GFP molecules from the native state through their complex energy landscape into the completely unfolded state. Unlike many smaller proteins, mechanical GFP unfolding proceeds by means of two subsequent intermediate states. The transition from the native state to the first intermediate state occurs near thermal equilibrium at approximate to35 pN and is characterized by detachment of a seven-residue N-terminal alpha-helix from the beta barrel. We measure the equilibrium free energy cost associated with this transition as 22 k(B)T. Detachment of this small a-helix completely destabilizes GFP thermodynamically even though the beta-barrel is still intact and can bear load. Mechanical stability of the protein on the millisecond timescale, however, is determined by the activation barrier of unfolding the beta-barrel out of this thermodynamically unstable intermediate state. High bandwidth, time-resolved measurements of the cantilever relaxation phase upon unfolding of the beta-barrel revealed a second metastable mechanical intermediate with one complete beta-strand detached from the barrel. Quantitative analysis of force distributions and lifetimes lead to a detailed picture of the complex mechanical unfolding pathway through a rough energy landscape.