Detecting structural changes in protein: polysaccharide complexes; an enabling technology.

能够检测相互作用的生物大分子的结构变化是理解复杂生物系统的基础，这既具有其固有的科学价值，也能够为生物技术和医学目的进行开发。目前，这涉及几种光谱技术：核磁共振、圆二色谱(CD)和红外光谱(FTIR)。然而，它们的成功依赖于选择性地观察到只有一个大分子成分的信号(如在核磁共振中)或来自信号去卷积的信号。后者假定每一种成分的光谱不受其相互接触的影响。虽然蛋白质-低聚糖的相互作用已被核磁共振广泛研究，但蛋白质与生物相关的多糖之间的相互作用却不能用核磁共振来研究，因为多糖不能移动和谱线加宽。此外，许多具有重要生物学意义的多糖，如糖胺聚糖(GAG)、透明质酸、软骨素、硫酸肝素和肝素，都含有对蛋白质二级结构分析所用光谱区域有贡献的基团。它们的光谱特征随环境的不同而变化，不能减去或去卷积。在BBSRC最近资助的一个项目(BB/D020794/1)中，我们首次表明，VCD(振动CD；红外线中的CD)选择性地检测Gag多糖溶液络合物中的蛋白质二级结构变化。实验是在使用常规红外光源的实验室FTIR仪器上进行的。限制VCD广泛应用于许多其他相互作用的因素是VCD信号的弱点(VCD信号的1-10%)需要大量蛋白质。我们将开发一种基于成熟的等离子体技术的台式光源，适用于产生比传统光源强度高得多的宽带IR，这也可以用于其他应用(例如真空UV中的CD)。该项目涉及利物浦大学和米德兰的精密工程公司M.I.Engineering的合作，这两家公司正寻求在科学仪器市场实现多元化。光源设计背后的原则(J.Phys.Chem.已经确立了使用VCD来选择性地检测复合体中的蛋白质结构变化(JACS130，(2008)2138)，从而将风险降至最低。此外，该公司还投入了时间进行讨论，并提供了全套工程图纸。该项目将需要建造和组装一台工作的VCD仪器，包括将信号源与商业VCD仪器相结合，并在对淀粉样蛋白：GAG复合体进行第一次实验测量之前为其性能建立基准。长期的商业目标是创造一种适销对路的产品，并设想该技术随后的几个应用。该仪器将能够选择性地检测目前难解的络合物，并在蛋白质组学、糖组学和系统生物学中得到应用。案例奖将为学生提供一段时间的科学和工程界面培训，涵盖整个仪器制造过程、安装、基准测试、操作优化、进行首次实验测量和研究复合体中的蛋白质结构。学生将在学术环境和工业和商业运营方面接受广泛的多学科培训，包括项目管理、商业战略、对科学仪器市场和驱动因素的了解，以及更广泛的工业应用、市场分析、财务方面(包括过程、开发成本和现金流动态)，并将掌握多学科可转移技能，并对如何优化未来的学术与产业合作有广泛的理解。

The ability to detect structural changes in interacting biological macromolecules underpins the quest to understand complex biological systems, both for its inherent scientific value and to enable exploitation for biotechnological and medical purposes. Currently, this involves several spectroscopic techniques; NMR, circular dichroism (CD) and infra red (FTIR). However, their success relies on selectively observing signals from only one macromolecular component (as in NMR) or from signal deconvolution. The latter presumes that the spectrum of each component is not affected by its contact with the other. While protein-oligosaccharide interactions are widely studied by NMR, those between proteins and biologically relevant polysaccharides cannot be, because of polysaccharide immobility and line broadening. Furthermore, many biologically important polysaccharides e.g. glycosaminoglycans (GAGs); hyaluronate, chondroitin, heparan sulfate and heparin contain groups which contribute to the spectrum in those spectral regions used for the analysis of protein secondary structure. Their spectral features change depending on their environment and cannot be subtracted or de-convoluted. During a recent BBSRC funded project (BB/D020794/1) we showed for the first time that, uniquely, VCD (vibrational CD; CD in the infra-red) selectively detects protein secondary structural changes in solution complexes of GAG polysaccharides. Experiments were conducted on a laboratory FTIR instrument using a conventional IR light source. The factor limiting the widespread application of VCD to a host of other interactions is the weaknesses of the VCD signal (1-10 % that of CD) requiring large amounts of protein. We will develop a bench-top light source based on established plasma technology, suitable for the generation of broad band IR of a considerably higher intensity than conventional light sources, which could also be exploited in other applications (e.g. CD in the vacuum UV). The project involves a collaboration between the University of Liverpool and a Midlands based precision engineering firm, M.I.Engineering, who are seeking to diversify into the scientific instrument market. The principle behind the light source design (J.Phys.Chem. 1984, 88, 488-490; Appl.Optics 46, (2007) 4948-4953), as well as the use of VCD to detect selectively protein structural changes in complexes (JACS 130, (2008) 2138) are already established, thereby minimising the risk. In addition, the company has also committed time to discussions and provided a full set of engineering drawings. The project will entail the construction and assembly of a working VCD instrument involving marrying the source with a commercial VCD instrument and establishing benchmarks for its performance before making the first experimental measurements on amyloid:GAG complexes. The long term business aim is to create a marketable product and several subsequent applications for the technology are envisaged. The instrument will enable the selective detection of currently refractory complexes and has applications in proteomics, glycomics and systems biology. The CASE award will provide the student with periods of training at the interface of science and engineering, covering the entire instrument building process, installation, benchmarking, operational optimization, making the first experimental measurements and investigating protein structure in complexes. The student will receive extensive multidisciplinary training in both the academic environment and the industrial and commercial operations including project management, business strategy, gain an appreciation of the scientific instrument market and drivers, as well as wider industrial applications, market analysis, financial aspects including process, development costs and cash-flow dynamics and will emerge equipped with multidisciplinary transferable skills and broad understanding of how to optimize future academic-industrial collaborations.