New strategies for spin labelling cysteine-rich proteins.

结构是功能的基础，这句格言在任何尺度上都是正确的。然而，探测纳米长度的结构可能是困难的，但对于了解结构并因此充分了解蛋白质的功能是必要的。测量蛋白质纳米距离的一种方法是使用电子顺磁共振（EPR）光谱学，它可以精确地测量分子对之间的偶极子-偶极子相互作用，就像条形磁铁之间的吸引力越近越强一样。为了使这种方法起作用，分子必须含有称为自由基的磁性物质。然而，它们在蛋白质中并不常见，因此必须在特定的、预先确定的位置添加到结构中。这些位点通常是特别活泼的氨基酸，叫做半胱氨酸。这个过程被称为自旋标记。在细胞外起作用的蛋白质通常含有很少的半胱氨酸，这使得它们在感兴趣的点上有这种氨基酸，然后被特异性地自旋标记。然而，在细胞内起作用的蛋白质可能含有许多半胱氨酸，如果将蛋白质分离并与自旋标签混合，则会有许多标签附着-这是EPR距离测量技术的缺点，该技术在最简单的一对自由基情况下是最准确的。此外，能够在活细胞内特异性标记蛋白质以测量它们之间的相互作用将是有用的，然而，如果注射半胱氨酸活性标记，它们将标记所有含半胱氨酸的蛋白质，而不仅仅是感兴趣的蛋白质。这些原则和问题延伸到需要对分离的蛋白质或活生物体内的蛋白质进行特定标记的各种技术。这里提出的工作旨在系统地探索一些自旋标记非天然氨基酸的选择——这些氨基酸被设计成具有特定的反应活性，可以在活细胞中被插入蛋白质中。2001年诺贝尔奖得主b·k·夏普勒斯和2010年诺贝尔奖得主a·铃木开发的尖端化学反应将被利用。由于EPR距离测量的准确性在蛋白质主链和标签本身之间的连接体较长时受到影响，这可能是有效结合非天然氨基酸所必需的，我们将开发既可以偶联到非天然氨基酸又可以偶联到邻近的天然氨基酸的自旋标签。这将减少自旋标签位置的不确定性。从中吸取的经验教训将用于测试是否有可能对天然氨基酸对进行特异性标记-例如，在富含半胱氨酸的蛋白质中，相邻的半胱氨酸对是否可以进行特异性标记？这个为期一年的项目将为高效和位点特异性自旋标记蛋白质奠定基础，无论它们是否含有多种半胱氨酸。此外，这项工作将为任何需要标记或标记的技术开发更通用的化学修饰非天然氨基酸的技术。这将对一系列学术、商业和医疗技术产生广泛的影响。

The maxim that Structure Underlies Function is true at any scale. However, probing structure over nanometre lengths can be difficult and yet is necessary to understand the structure and therefore fully understand the function of proteins. One method for measuring nanometre distances on proteins is by using electron paramagnetic resonance (EPR) spectroscopy which can accurately measure the dipole-dipole interaction between pairs of molecules, in a similar way that the attraction between bar magnets feels stronger as the two get closer together. In order for this method to work, the molecules must contain magnetic species called radicals. However, these are not very common in proteins and so they must be added to the structure at particular, pre-determined, positions. Most commonly these sites are particularly reactive amino acids called cysteines. This process is called spin labelling. Proteins which function outside a cell usually contain very few cysteines which allows them to have this amino acid engineered in at points of interest, and so then be specifically spin-labelled.However, proteins that function within the cell may contain many cysteines and if the protein were isolated and mixed with spin label there would be lots of labels attached - a disadvantage for the EPR distance measurement technique which is most accurate in the simplest case of a pair of radicals. Additionally, it would be useful to be able to label proteins specifically within a living cell to enable measurements of their interactions there, however if cysteine reactive labels were injected they would label all cysteine-containing proteins - not just the one of interest. These principles and problems extend to all sorts of techniques that require specific labelling of isolated proteins or proteins within a living organism. The work proposed here seeks to systematically explore some of the options for spin labelling unnatural amino acids - these are amino acids that have been designed with specific reactivities and can be inserted into proteins as they are made in a living cell. Cutting-edge chemical reactions such those developed by 2001 Nobel prize winner B. K. Sharpless and 2010 Nobel prize winner A. Suzuki, shall be utilised. Since the accuracy of the EPR distance measurements is affected when the linker between the backbone of the protein and the label itself is long, and this may be necessary for efficient incorporation of the unnatural amino acid, we shall develop spin labels that can be coupled both to the unnatural amino acid and a natural amino acid adjacent to it. This will reduce the uncertainty in the position of the spin label. The lessons learned from this will be used to test whether it might be possible to specifically label pairs of natural amino acids - e.g. can pairs of adjacent cysteines be labelled specifically in a cysteine-rich protein? This one-year project will lay the groundwork for efficiently and site-specifically spin labelling proteins, regardless of whether they contain multiple cysteines or not. Further, the work will develop the more general technique of chemically modifying unnatural amino acids for any technology that requires labelling or tagging. This will have wide-reaching impact on a range of academic, commercial and medical techniques.