Direct observation of ultrafast folding and denatured state dynamics in single protein molecules
Direct observation of ultrafast folding and denatured state dynamics in single protein molecules
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DOI：
10.1073/pnas.0910860106
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发表时间：
2009-11-03
影响因子：
11.1
通讯作者：
Fersht, Alan R.
中科院分区：
文献类型：
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作者：
Neuweiler, Hannes;Johnson, Christopher M.;Fersht, Alan R.
单分子荧光共振能量转移(SMFRET)实验在蛋白质折叠研究中是非常有用的，但通常被限制在大于100亩S和大于约2纳米的距离的时间尺度上。我们使用光诱导电子转移猝灭单分子荧光，检测短程事件，结合荧光相关光谱(PET-FCS)，以纳秒的时间分辨率研究了小结合域BBL的折叠动力学。折叠动力学在自相关函数中表现为10亩S衰变，这是由于单个分子的变性构象和天然构象之间的随机波动造成的。观察到的速率常数与探测器无关，并与传统的温度跳跃(T-JUMP)测量的值非常一致。在PET-FCS数据中检测到了亚微秒的松弛，该数据报告了在热变性状态下链内接触形成的动力学。我们设计了一个Bbl的突变体，它在有利于亲本野生型(“D-phys”)折叠的反应条件下进行变性。D-phys具有与热变性状态相同的动力学特征，并呈现分段扩散，其链内接触形成的时间常数为500 ns。这一时间常数比折叠快10倍以上，并在估计为折叠的“速度极限”范围内。D-PYS表现出与随机线圈的显著偏差。链内扩散的溶剂粘度和温度依赖性表明，分子内相互作用的存在抑制了链的运动。PET-FCS与蛋白质工程相结合是研究蛋白质超快折叠早期事件和机制的有力手段。
Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ("D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the "speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.