Molecular Mechanics of Enzymes

激光可用于执行多种角色，包括在激光阱（所谓的光镊）内感测、操纵和移动物体。值得注意的是，光的应用允许人们对单个蛋白质分子施加微小的光学力。通过拉动单个分子，可以非常详细地研究DNA和蛋白质结构。蛋白质的拉伸和展开揭示了赋予它们三维结构的分子内力。光镊实验也允许直接测量由单个马达蛋白施加的皮牛顿力。然而，光镊和其他单分子技术目前还不够灵敏，无法解析飞秒牛顿（fN）力，因此还不能研究所有的分子力。一个重要但迄今知之甚少的例子是活性酶在催化生命系统中的反应时所施加的微小fN力。这项研究计划将开发一种全新的、灵敏度更高的替代光镊技术。该计划中开发的纳米传感器将提供光学“手”，可以探测和探测酶的fN力。这使得精确的能量感测的构象变化的酶，即自己的变形运动，第一次。这种测量将提供基本的洞察力，推动催化所需的构象变化的力量。我们将可视化酶的运动，这将使我们能够开发更准确的模型来预测这些非常重要的分子机器如何运作。我们的方法将揭开自然界设计一类纳米机器的原理，这些纳米机器执行大多数重要的生物化学和分子信号，使我们的身体工作。该计划开发的技术不仅可以感知酶施加的力，还可以让我们操纵活性酶的复杂运动。这种控制提供了使酶中的某些分子组织模式比另一种更可能的可能性，并且可以用于控制酶活性。展示这一能力将为未来操纵和开发合成生物分子机制以及设计用于特定化学或医疗任务的酶奠定基础。我们的影响工作之路将展示我们的技术在医疗诊断测试中的极端敏感性，我们将为人类病原体开发。纳米传感器将通过附着酶进行修饰。这将使我们能够在人类真菌病原体白色念珠菌细胞壁上存在的不同糖的酶分解过程中测量病原体特异性信号-白色念珠菌是一种每年导致约25万例血流感染的致病真菌。这种测量将能够比目前基于细胞培养的感染微生物诊断更快速地鉴定真菌病原体。将对位于南非开普敦的AFGrica真菌免疫组提供的样本进行测试。

Laser light can be used to perform multiple roles, including sensing, manipulating and moving objects within a laser trap (so-called optical tweezers). It is remarkable that the application of light allows one to exert minute optical forces on individual protein molecules. By pulling on individual molecules it has been possible to study DNA and protein structures in great detail. The pulling and unfolding of proteins has revealed the intramolecular forces that give them their three-dimensional structure. Optical tweezer experiments have also allowed the direct measurement of pico-Newton forces that are exerted by individual motor proteins. However, optical tweezers and other single-molecule techniques are currently not sensitive enough to resolve femto-Newton (fN) forces, and hence not all molecular forces can yet be investigated. An important, but so far poorly understood example is the miniscule fN forces that are exerted by active enzymes when they are catalysing reactions in living systems.This research programme will develop an entirely novel and much more sensitive alternative optical tweezer technology. The nanosensors developed in this programme will provide optical 'hands' that can probe and feel-out fN forces of enzymes. This allows precise sensing of the energetics of conformational changes of enzymes, i.e. their own deforming motion, for the first time. Such measurements will provide fundamental insights into the forces that drive the conformational changes that are required for catalysis. We will visualise the enzyme movements and this will allow us to develop more accurate models to predict how these very important molecular machines function. Our approach will unravel nature's design principles for a class of nanomachines that carry out most of the important biochemistry and molecular signalling that make our bodies work. The technology developed in this programme will not only sense forces exerted by enzymes, but allows us to manipulate the complex motions of active enzymes. Such control offers the possibility of making some patterns of molecular organisation in an enzyme more likely than another, and can be used to control enzymatic activity. Demonstrating this capability will prepare the ground for future manipulation and exploitation of synthetic biomolecular machinery and designing enzymes for specific chemical or medical tasks.Our pathway to impact work will demonstrate the extreme sensitivity of our technology in healthcare diagnostic tests that we will develop for human pathogens. Nanosensors will be modified by attaching enzymes. This will allow us to measure pathogen-specific signals during enzymatic breakdown of different sugars present on cell-walls of the human fungal pathogen Candida albicans - a pathogenic fungus that causes around 250,000 blood stream infections per year. This measurement will enable a more rapid identification of fungal pathogens than current microbial diagnostics of infections based on cell cultures. This approach will be tested on samples provided by the Fungal Immunology Group, AFGrica, located in Cape Town, South Africa.