Developing and investigating an ultra-stable molecular hub for bionanotechnology

When biologists wish to isolate or immobilise an object (proteins, DNA, RNA, sugars, small organic molecules, nanoparticles), they will very often attach the vitamin biotin to this object, so that the object can be captured by the protein streptavidin. The link from biotin to streptavidin is one of the strongest known in nature, but this link can be broken in challenging situations, so limiting many biological experiments and devices. We have made an altered streptavidin which forms an even stronger link to biotin, so that biotin is captured for longer and stays bound in harsher conditions. We plan to determine at the atomic level why this new interaction is stronger. This insight should help to illuminate the basis of ultra-strong binding, and so assist the design of drugs that can bind more stably to their targets and thus be more effective. We will also engineer streptavidin so that it can anchor two different kinds of molecules, enabling biologists to create new assemblies to combine and control the functions of biomolecules. We ourselves will use these assemblies to explore how growth factors can either work together or compete in transmitting signals inside the cell, so determining whether the cell lives, dies, or divides.

The binding of biotin to streptavidin is one of the most widely used interactions in biological research and biotechnology. Streptavidin binds biotin-conjugates with exceptional stability, but dissociation does occur and can be a limitation in imaging, DNA amplification, and nanotechnology. We have identified a mutant streptavidin, which we call traptavidin, showing more than 10-fold slower biotin dissociation, increased mechanical strength, and improved thermostability: this resilience should find diverse applications in biomolecular analysis and assembly. We propose to use crystallography and isothermal titration calorimetry to determine the structural and energetic factors responsible for this tenacious protein-ligand interaction. This understanding should illuminate the intermolecular interactions and dynamics that can confer femtomolar affinity. Streptavidin has 4 biotin-binding sites of equal affinity but we have developed a way to control exactly how many of the biotin binding sites are active, so that harnessing the strength of streptavidin binding does not have to be accompanied by 4-way cross-linking. In this work we will develop tetramers that can stably bind 0-4 copies of two distinct ligands in a precisely defined orientation, so that streptavidin can act as a molecular hub for assembly in synthetic biology and bionanotechnology. We will assemble a hub that precisely orients two different tyrosine kinase receptors, to determine the spatial effects on cross-talk between two signalling pathways important for cell growth, differentiation and survival.

Who will benefit from this research? Development of orthogonal and single-chain streptavidin tetramers may be beneficial to a range of biotechnology companies, and also to pharmaceutical companies who perform protein-based research. Products developed by these companies could lead to enhanced therapies or diagnostic approaches and so be beneficial to the general public and the National Health Service. How will they benefit from this research? The new streptavidin variants may allow stable, simple and defined-orientation protein immobilisation in biosensors, columns for purification, and protein microarrays. Traptavidin may increase sensitivity in immunoassays and enhance the stability of tracking in microscopy. The new streptavidin variants may directly comprise part of a commercial product/kit, or facilitate research leading to the generation of other products. Understanding the cross-talk from EGFR will help to understand the effects of IGF1R in aging, diabetes and cardiovascular disease and so may contribute to manipulation of IGF1R in medicine, which is already a growing therapeutic target. The likely time-scale is for commercial licensing of these new streptavidin variants is in the second half of 2013. This project will provide important training for the postdoctoral researcher in developing and executing a project which generates new tools and applies them in cell biology. The researcher will be exposed to a wide range of techniques, many of which are likely to be new, including quantitative fluorescent microscopy, crystallography and structure-based protein engineering. The postdoctoral researcher will be supported in the development of their presentation skills and career development by presenting their work within the university and at national and international conferences and by interacting with the media, whenever possible, following successful completion of a milestone. What will be done to ensure that they benefit from this research? Publication in high impact peer-reviewed international journals is the most effective way for us to communicate our findings to potential industrial partners, as well as to fellow academics. Similarly, many of the conferences that the PI attends in chemical biology, proteomics and biophysics contain presenters and audience members from both industry and academia. At this stage we have not engaged with beneficiaries but Isis Innovation are beginning negotiations based on our traptavidin patent. We will follow the same route for protection and dissemination of new streptavidin variants arising from this proposal. Isis Innovation has substantial experience in licensing and links to many UK and international companies but may well establish new links. The PI has previously attended 2 meetings of the Sensors and Instrumentation Knowledge Centre and my laboratory members have presented posters here. The PI will seek to give a talk at a future meeting of this network. As we achieve key results in this project we will communicate with the University of Oxford press office and the BBSRC to ensure that significant findings are communicated widely, to the general public locally and nationally. We will publish detailed protocols to facilitate adoption of the streptavidin variants by other users, as we have done previously for the acceptor peptide and monovalent streptavidin in Nature Protocols. We will also provide rapid e-mail feedback as we have done for the laboratories using biotin ligase and monovalent streptavidin for diverse applications.