A quantitative biomolecular interaction suite

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

Proteins in living organisms frequently interact with each other and with DNA, RNA and small molecules. In order to be able to understand how all these interactions help an organisms to survive and build models for these interactions, it is important to be able to measure the interactions in a quantitative way. In this proposal we will establish a facility which will house two separate machines which use different biophysical methods to accurately measure interactions between molecules found in biology. We will use these machines to carry out a wide variety of research ranging from how complexes of proteins assemble to how the walls of bacterial cells work.

Technical Summary

The accurate quantitative measurement of biomolecular interactions is an important aspect of modern molecular biology research. Such quantitation will ultimately allow us to build accurate models for a variety of biological process. In this proposal we will establish a facility which makes use of two distinct biophysical techniques to quantitate biomolecular interactions: 1) isothermal titration calorimetry (ITC) and 2) microscale thermophoresis (MT). The facility will chiefly comprise of a Microcal ItC200 machine and a Nanotemper Monolith NT.115 machine. These two machines each have distinctive features and the combination of the two biophysical techniques they employ will provide a world class facility for biomolecular interaction analysis. The facility will be open to both internal and external users and will allow biomolecular interaction quantification using completely unlabelled samples in solution (using ITC). Alternatively using MT, investigators will be able to measure biomolecular interactions in complex mixtures such as cell lysates, where one of the biomolecules is fluorescently labelled. The exquisite sensitivity of MT means that quantitative measurements will be accessible even for very large proteins or complexes, which are frequently difficult to purify in sufficient quantities for ITC work. The applicants will utilise this facility to quantitate a wide range of biomolecular interactions including: the analysis of TREX mRNA complex assembly, membrane trafficking, nucleases, bacterial cell wall structure, actin binding proteins, novel protein linking technologies, cAMP and nitric oxide signalling, protein secretion, carbon monoxide releasing molecules and defense mechanisms of bacterial pathogens.

Planned Impact

(A) Potential Beneficiaries
Beneficiaries of the research will be academics, health professionals, industry and schools.
(B) How might they benefit ?
(i) Academic Beneficiaries will be post-doctoral researchers, PhD students and technicians from a wide range of disciplines including biochemists, through cell biologists to systems biologists, chemists and molecular biologists. They will receive high quality training from the PIs and technicians associated with this facility. They will also publish high quality high impact publications including reviews. Reviews will ensure coverage not just to those in the immediate field, but to a broader audience of biologists at a range of academic levels. Work at this level enhances the reputation of UK science in general and this is key to confidence in the competitiveness of UK, which is directly related to wealth and economic output of the higher education industry. Timescale 1-5 years.
(ii) Health related disciplines will benefit from this study. The quantitative studies proposed in this project will provide a better understanding of the biomolecular interactions which impinge on a range of human disorders and this provides the opportunity to impact on the treatment of these disorders in the future. These conditions include: Neuromuscular and neurodegenerative disorders, cancer, microbial infections, chronic pain and acromegaly. Timescale 2-5 years.
(iii) Industry. Typically drugs which target the range of diseases and infections described above require small molecule inhibitors. This project will allow us to make detailed quantitative measurements of small molecule interactions with other biomolecules and such measurements will impact on the design in the pharmaceutical industry of suitable drugs for treatment of such disorders and infections. Small molecules which associate with biomolecules might also be used in the biotechnology, agriculture and engineering industries and the work proposed here with this facility will aid in the design of such useful molecules for these industries.
In addition the post-docs, PhD students and technicians trained in the laboratories involved may well enter industry and carry out much of the research and development in the future. Our ability to supply highly trained scientists will positively impact on the ability of the UK industrial sector to remain competitive. Timescale 2-5 years.
(iv) Schools. The future of science depends on enthusiastic young scientists. The best way to achieve this is to provide stimulating scientific based activities for school children. Several of the applicants are involved in visiting schools to give talks and run activities. Timescale: Schools are visited on an annual basis.
 
Description 1. Showed the formation of a complex between two members of the AAA+ super family of motor proteins is mediated by interactions with two essential arginines in the protein respective AAA+ domains. The MST complimented single molecule spectroscopy of the complexes.
2. Revealed the precise molecular basis for hypophosphite specificity and the reporting of a novel P-H...Pi bond, unreported in the literature for proteins. MST was used to determine the Kd of hypophosphite and other ligands binding to a variety of phosphite binding proteins.
3. MST has been used to characterize the affinity between ChlD and a series of mutants of ChlH. This study pieces together the protein-protein interactions that occur within the magnesium chelatase complex to power the a reaction from a motor several nm away from the active site of metal ion insertion into porphyrin.
4. We have studied the non-specific binding interactions that occur during digestion in mammals. This includes tannin and phytate binding to proteins. Both tannin and phytate are present in grain and other vegetable foods. They both bind to enzymes and protein substrates interfering with digestion and are considered anti-nutritional. The ultimate aim of this research is to make animal feed for chickens and pigs give better yields (kg meat/kg feed). So far we have developed the ITC assay for the binding interaction and the enzyme assay for phytase activity.
5. Mutation of arginine residues lying within the polyproline region of the yeast WASP homologue reduces binding affinity for binding directly to actin. This work is helping us to understand the mechanism of WASP family proteins in Arp2/3-independent actin nucleation.
Exploitation Route Our findings may help with with feeding livestock and provide new ways to harvets energy by manipulation of photosynthetic bacteria.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Security and Diplomacy