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

在相互作用的生物大分子中检测结构变化的能力，支持了对复杂生物系统的理解，这既是为了其固有的科学价值，也是为了生物技术和医学目的的开发。目前，这涉及到几种光谱技术；核磁共振，圆二色性（CD）和红外（FTIR）。然而，他们的成功依赖于有选择地观察来自单一大分子成分（如核磁共振）或信号反卷积的信号。后者假定每个组分的光谱不受其与其他组分接触的影响。虽然蛋白质与低聚糖的相互作用已被核磁共振广泛研究，但由于多糖的不动性和谱线拓宽，蛋白质与生物相关多糖之间的相互作用无法被研究。此外，许多具有重要生物学意义的多糖，如糖胺聚糖（GAGs）；透明质酸、软骨素、硫酸肝素和肝素在用于分析蛋白质二级结构的光谱区域中含有对光谱有贡献的基团。它们的光谱特征随环境的变化而变化，不能被减除或去卷积。在最近的一个BBSRC资助的项目（BB/D020794/1）中，我们首次展示了VCD（振动CD；红外CD）选择性地检测GAG多糖溶液配合物中蛋白质二级结构的变化。实验采用常规红外光源，在实验室红外仪器上进行。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（CD信号的1- 10%）需要大量的蛋白质。我们将开发一种基于已建立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红外，这也可以用于其他应用（例如真空紫外中的CD）。该项目涉及利物浦大学和位于米德兰兹的精密工程公司mii engineering之间的合作，后者正在寻求进入科学仪器市场的多元化。光源设计的原理（j .物理化学）。1984, 88, 488-490；达成。光学46，（2007）4948-4953)，以及使用VCD选择性检测复合物中的蛋白质结构变化（JACS 130,(2008) 2138）已经建立，从而将风险降至最低。此外，该公司还抽出时间进行讨论，并提供了全套工程图纸。该项目将需要构建和组装一个工作VCD仪器，包括将源与商用VCD仪器结合起来，并在对淀粉样蛋白：GAG复合物进行第一次实验测量之前建立其性能基准。长期的商业目标是创造一种可销售的产品，并设想了该技术的几个后续应用。该仪器将能够选择性检测当前难降解复合物，并在蛋白质组学，糖组学和系统生物学中得到应用。CASE奖项将为学生提供科学和工程界面的培训，涵盖整个仪器建造过程、安装、基准测试、操作优化、进行首次实验测量和研究复合物中的蛋白质结构。学生将接受广泛的多学科培训，包括学术环境和工业和商业运作，包括项目管理，商业战略，了解科学仪器市场和驱动因素，以及更广泛的工业应用，市场分析，财务方面，包括流程，开发成本和现金流动态，并将配备多学科的可转移技能和如何优化未来的学术-工业合作的广泛理解。

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.