Catching a protein in the act.
Catching a protein in the act.
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在行动中捕获蛋白质。
DOI：
10.1073/pnas.0914486107
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发表时间：
2010
影响因子：
11.1
通讯作者：
Hummer,Gerhard
中科院分区：
文献类型：
--
作者：
Hummer,Gerhard
眼见为实。这句老话总结了生物学和生物物理学的主要目标之一：观察并最终了解维持生命的分子过程。由于实验和模拟的进步，我们正在迅速获得对关键生物分子过程的分子水平的理解。然而，实验仍然主要限于中间体的鉴定和表征沿着反应路径，需要大量的人口和足够长的寿命是可检测的。最终，对分子机制的充分理解还需要表征中间体之间的过渡。人们甚至可能会说，分子机制包含在分离中间体的过渡态中。但是，尽管最近取得了许多进展[例如，通过荧光光谱确定了蛋白质折叠的过渡时间上限（1）]，但我们对生物分子过渡的机械理解主要来自间接测量，例如φ值分析（2），使过渡本身在很大程度上处于黑暗中。在本期PNAS中，Vreede et al. (3)展示了现代分子模拟技术如何用于阐明分子跃迁，即使是那些发生在毫秒级的时间尺度上，远远超过标准分子动力学计算的分子跃迁。Vreede等人(3)探索光敏黄蛋白（PYP）光诱导构象变化序列中的关键步骤。PYP是一种光受体，被认为是由细菌Halorhodospira halophila用来检测并最终避免蓝光。PYP的疏水性核心中填充了一个共价连接的发色团，它在吸收光后发生反式至顺式异构化（图1A）。作为第一个主要的中间体，pR状态在纳秒时间尺度上形成。在pR中，发色团与其芳环的紧密堆积限制了响应于光吸收的初始构象变化的程度（7）。在大约500 μs的时间尺度上，一个质子从Glu-46内部转移到发色团，形成pB′中间体。几毫秒后（4），由反式-顺式异构化建立的机械应力被释放，在所得pB中间体中产生蛋白质结构的显著变化，包括部分解折叠（8）。从pB′到pB的转变是Vreede等人模拟的重点。（三）、Vreede等人怎么能。(3)使用分子模拟，每个只覆盖几纳秒，研究一个过程，发生在毫秒的时间尺度？诀窍在于，在这毫秒的时间里，每一个PYP蛋白主要是等待一系列随机的热反冲，将pB′状态转换为pB。但是，单个PYP分子从pB′到pB的实际跃迁可能比相互转化的速率快许多个数量级（图1B）。然后，目标变成仅对连接反应物和产物状态的相对快速的跃迁路径进行采样，避免反应物状态pB’中的长等待时间。这样的反应轨迹的集合可以通过transitionpath采样（5，6）有效地创建。在分子动力学模拟中，分子系统（包括溶剂）的相互作用由能量函数表示，该能量函数包括键合、静电和货车范德华相互作用。系统在这个能量面上按照经典的牛顿动力学运动。在过渡路径采样（5，6）中，反应轨迹被创建为与非常长的平衡模拟中完全相同的分布。配置空间被划分为...
Seeing is believing. This old saying sums up one of the main goals in biology and biophysics: to watch and ultimately understand the molecular processes that sustain life. Thanks to advances in both experimentation and simulation, we are rapidly gaining a molecular-level understanding of key biomolecular processes. Nevertheless, experiments remain largely limited to the identification and characterization of intermediates along a reaction path, requiring significant populations and sufficiently long lifetimes to be detectable. Ultimately, a full understanding of the molecular mechanisms will also require the characterization of the transitions between the intermediates. One might even argue that the molecular mechanism is contained in the transition state separating the intermediates. But despite much recent progress [eg, with an upper bound determined for the transition time in protein folding from fluorescence spectroscopy (1)], our mechanistic understanding of biomolecular transitions is derived primarily from indirect measurements, such as phi-value analysis (2), leaving the transitions themselves largely in the dark. In this issue of the PNAS, Vreede et al.(3) show how modern molecular simulation techniques can be used to shed light on molecular transitions, even those occurring on a millisecond timescale far beyond that of standard molecular dynamics calculations. Vreede et al.(3) explore a key step in the sequence of light-induced conformational changes of photoactive yellow protein (PYP). PYP is a light receptor thought to be used by the bacterium Halorhodospira halophila to detect and ultimately avoid blue light. Packed into the hydrophobic core of PYP is a covalently attached chromophore that undergoes a trans-to-cis isomerization upon absorption of light (Fig. 1A). As the first major intermediate, the pR state forms on a nanosecond timescale. In pR, the tight packing of the chromophore with its aromatic ring limits the extent of the initial conformational changes in response to photoabsorption (7). On a timescale of≈ 500 μs (4), a proton is internally transferred from Glu-46 to the chromophore to form the pB′ intermediate. After a few milliseconds (4), the mechanical stress built up by the trans-cis isomerization is relieved, producing dramatic changes of the protein structure in the resulting pB intermediate, including partial unfolding (8). This transition from pB′ to pB is the focus of the simulations by Vreede et al.(3). How can Vreede et al.(3) use molecular simulations, each covering only a few nanoseconds, to study a process that occurs on a timescale of milliseconds? The trick is the realization that during this millisecond time, each individual PYP protein is primarily just waiting around for a series of random thermal kicks that convert the pB′ state to pB. But the actual transition of an individual PYP molecule from pB′ to pB can be many orders of magnitude faster than the rate of interconversion (1)(Fig. 1B). The goal then becomes to sample only the relatively fast transition paths that connect the reactant and product states, avoiding the long waiting times in the reactant state pB′. An ensemble of such reactive trajectories can be created efficiently by transitionpath sampling (5, 6). In molecular dynamics simulations, the interactions of the molecular system, including the solvent, are represented by an energy function that includes terms for bonding, electrostatics, and van der Waals interactions. The system moves on this energy surface according to classical Newtonian dynamics. In transition-path sampling (5, 6), reactive trajectories are created with exactly the same distribution as in very long equilibrium simulations. Configuration space is divided …