Long-term tracking of protein conformational dynamics through single-molecule thermal snapshot spectroscopy

来自X射线晶体学的蛋白质结构是详细但冻结的图像：它们缺乏关于蛋白质如何波动，适应和结合其他生物分子的信息，以及执行其功能所涉及的运动。同样，标准单分子光谱非常适合研究室温下单个蛋白质的行为，但在时间分辨率和最大观察时间方面都存在固有的限制。本申请提出了一种在单分子水平上访问蛋白质动力学的通用方法。其中心思想是在单个生物分子自然运动的不同阶段反复冷冻猝灭，提供其动力学的定格电影。这将通过快速（微秒）冷冻和解冻循环来实现，在低温下运动被冻结，在高温下运动自然进行。这种方法具有单分子观测的所有优点：它消除了系综平均，并且不需要任何同步，因为每个单分子在任何时候都处于单一状态。而其他技术，例如，冷冻电子显微镜，揭示了在单个分子的集合体中发现的构象的传播，这种方法将记录构象的序列，提供对势能景观中的途径的独特见解。Orrit的实验室已经证明了在寒冷环境中通过激光加热实现微秒级温度循环的可行性。该提案设定了两个目标，将这些探索性实验转化为可靠的实验方法：a）将温度循环方法应用于其动力学特性良好的生物分子，从而证明该方法的可靠性;B）与蛋白质动力学研究领域的世界级团体合作，将该技术应用于金属蛋白和腺苷酸激酶的蛋白质动力学中的开放问题。

Protein structures from X-ray crystallography are detailed but frozen images: they lack information about how proteins fluctuate, adapt and bind to other biomolecules, and which movements are involved in performing their functions. Similarly, standard single-molecule spectroscopy is well suited to studying the behaviour of individual proteins at room temperature, but is inherently limited, both in its time resolution and in maximum observation times. This application proposes a general method to access protein dynamics at the single-molecule level. The central idea is to freeze-quench a single biomolecule repeatedly at different stages of its natural motion, providing a stop-motion movie of its dynamics. This will be achieved through fast (microsecond) freezing and thawing cycles, between a low temperature where motion is frozen and a high temperature where motion proceeds naturally. This method holds all the advantages of single-molecule observations: it eliminates ensemble averaging and does not require any synchronization, for each single molecule is in a single state at any time. Whereas other techniques, e.g., cryo-electron microscopy, do reveal the spread of conformations found in ensembles of single molecules, this method will record the sequence of conformations, providing unique insight into pathways in the potential energy landscape.Initial experiments in M. Orrit's lab have demonstrated the feasibility of microsecond temperature cycles by means of laser heating in a cold environment. This proposal sets two objectives to turn these exploratory experiments into a reliable experimental method:a) to apply the temperature cycle method to biomolecules whose dynamics is well characterized, and thereby to provide proof of the reliability of the method;b) to apply the technique to open problems in protein dynamics on metallo-proteins and adenylate kinase, in collaboration with world-class groups in the field of protein dynamics research.