Optical spectroscopy for biomolecular interactions

申请人希望购买总计139,710英镑的光谱设备（包括服务合同），以实现荧光检测线性二色测量（FDLD），并使用荧光和吸收数据解释FDLD数据。光谱仪还将用于线性二色性（LD）、圆二色性（CD）、荧光、荧光偏振各向异性和吸光度测量，作为独立实验，以及与FDLD新技术结合使用。Jasco UK Ltd.为该项目贡献了49,219英镑，其中6,000英镑的员工时间用于在华威大学进行培训和支持研讨会，其余部分用于购买仪器的折扣。雅斯科希望将华威建立为雅斯科偏振光谱学卓越中心，这将进一步加强申请人与雅斯科英国有限公司之间的合作。Crystal Precision Optics将贡献4000英镑的员工时间来支持该项目产生的新设备的设计和开发。将需要少量的PI时间来监督采购、安装和将设备建立为多用户设施。基本原理：所有的生物过程基本上都是分子之间的相互作用，主要是大分子之间或大分子与小分子之间的相互作用。尽管我们在描述这种相互作用的能力和正在发生的“单分子”革命方面取得了重大进展，但我们仍然难以测量分子相互作用的关键特性。对于一些重要的生物分子来说尤其如此，这些生物分子已经被证明很难用现有的结构技术（如晶体学和核磁共振）来研究。这些包括长聚合结构：DNA；DNA-ligand情结;纤维，包括纤维蛋白；还有膜蛋白。希望探索相互作用的时间依赖性，包括酶动力学，纤维组装或蛋白质插入膜，引入了一个额外的复杂性维度。本提案的目的是建立FDLD的新发明技术，使英国社区可以从其优势中受益。核心FDLD仪器将得到标准荧光和吸收仪器的支持，以便进行数据解释。这三种仪器还将用于独立的LD、CD、荧光、偏振荧光和相互作用的生物分子系统的测量。在过去的几年里，华威大学已经成为国内乃至国际上基于流动线性二色性（LD）技术的创新中心，这是平行和垂直于方向轴的光偏振的吸光度差异。LD可用于推断大范围系统的动力学和结构信息，唯一的要求是它们具有吸光度光谱并且可以定向。流动LD要求样品足够长，以便在溶液中通过剪切力定向，并且非常适合DNA和纤维蛋白。我们在定向膜系统方面也取得了重大成功，因为脂质体的流动扭曲产生了一个定向轴。在配体结合的背景下，LD的主要吸引力在于它仅对与长系统结合的分子具有选择性。因此，例如，仅检测PCR产物，而不检测游离核苷酸的背景种群。我们开发的新技术之一是荧光检测线性二色性（FDLD）。这是当前应用程序的重点。FDLD抓住了(i) LD（即仅检测定向样品）和（ii）荧光（即仅检测荧光团并且比基于吸收的技术灵敏度更高）的分子间相互作用的优点。在FDLD的概念证明论文中，我们展示了如何选择性地使用FDLD来研究光谱位于DNA吸光度带下的配体在DNA上的取向。

The applicants wish to purchase spectroscopic equipment totalling £139,710 (including service contracts) to enable fluorescence detected linear dichroism measurements (FDLD) and the interpretation of the FDLD data using fluorescence and absorption data. The spectrometers will also be used for linear dichroism (LD), circular dichroism (CD), fluorescence, fluorescence polarisation anisotropy and absorbance measurements as stand-alone experiments as well as in conjunction with the new technique of FDLD. Jasco UK Ltd. are contributing £49,219 to the project in the form of £6,000 of staff time for training and supporting workshops to be run at Warwick and the remainder in discounts on the purchase of the instruments. Jasco wish to establish Warwick as the Jasco Centre of excellence in Polarised Spectroscopy which will further enhance the collaboration between the applicants and Jasco UK Ltd. Crystal Precision Optics will contribute £4,000 of staff time to support design and development of new equipment emanating from this project. A small amount of PI time will be required to oversee the procurement, installation, and establishing the equipment as a multi-user facility. Rationale underlying proposal: All biological processes are fundamentally interactions between molecules, mainly between macromolecules or between macromolecules and small molecules. Despite significant advances in our ability to characterise such interactions and the 'single-molecule' revolution that is taking place, we still struggle to measure key properties of molecular interactions. This is particularly true for the significant classes of biological molecules that have proved difficult to study by established structural techniques such as crystallography and NMR. These include long polymeric structures: DNA; DNA-ligand complexes; fibres including fibrous proteins; and also membrane proteins. Wishing to probe the time dependence of interactions including enzyme kinetics, fibre assembly or protein insertion into membranes introduces an added dimension of complexity. The aim of this proposal is to establish the newly invented technique of FDLD so the UK community can benefit from its advantages. The core FDLD instrument will be supported by standard fluorescence and absorption instruments to enable data interpretation. All three instruments will also be used for independent LD, CD, fluorescence, polarised fluorescence and measurements of interacting biomolecular systems. Over the last few years Warwick has become the national and indeed international hub for innovation based on the technique of flow linear dichroism (LD), which is the difference in absorbance of light polarised parallel and perpendicular to an orientation axis. LD can be used to deduce kinetic and structural information about a wide range of systems, the only requirements being that they have absorbance spectroscopy and they can be oriented. Flow LD requires samples to be long enough to be oriented by shear forces in solution and is ideally suited to DNA and fibrous proteins. We have also had significant success in orienting membrane systems as the flow-distortion of liposomes creates an orientation axis. In the context of ligand binding, the key attraction of LD is that it is selective only for those molecules bound to the long system. Thus, e.g., only PCR products are detected, not the background population of free nucleotides. One of the new techniques we have developed is fluorescence detected linear dichroism (FDLD). This is the focus of the current application. FDLD captures the advantages for intermolecular interactions of (i) LD (namely detecting only oriented samples) and (ii) fluorescence (namely only detecting fluorophores and with higher sensitivity than absorption-based techniques). In the proof of concept paper for FDLD we showed how FDLD can be used selectively to study the orientation on DNA of ligands whose spectroscopy lies under the DNA absorbance bands.