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

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences


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.


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