It is clear that very weak protein–protein interactions can be extremely important in various areas of biology1. In many systems transient and reversible protein binding can be vital, these systems include such things as electron-transport, replication, transcription, cell signalling and some aspects of catalysis. Protein–protein interactions can exhibit a broad range of affinities ranging from very tightly bond complexes having sub-nanomolar affinities to very weak or transient interactions that can have affinities in the millimolar range. A number of accurate and well tested methods have been developed to study and measurement of high affinity binding but there is considerable difficulty in measuring and characterising very low affinity binding2. Perhaps the biggest problem in characterising low affinity binders is the issue of avoiding complications caused by the contributions from unbound protein.
Recent developments in spin-labelling technologies, especially those using copper 2+ ions, have opened up a variety of new opportunities for using electron paramagnetic resonance (EPR) to probe and measure biomolecular complexes3. In this project the application of copper ion based spin labels, along with the more traditional nitroxide spin labels will be investigated with the aim of developing methodologies to measure weak binding protein-protein complexes.
EPR of spin labelled proteins can be used to measure the conformation and binding arrangement of a pair of interacting proteins (by measuring long distances) however this is has only been possible in situations where the affinity of binding is strong. When binding affinity is low then the ratio of bound to unbound protein will be low. This has two effects, one is that the concentration of bound complex will be low causing difficulties related to the sensitivity of the detection and the other complication is that the presence of spin-labelled but unbound protein will, in itself, severely diminish the signal to noise ratio.
We have recently shown that a novel system, that allows us to place a copper ion site specifically on a proteins secondary structure, coupled with more traditional nitroxide spin labelling on a cysteine residue can give spectacular results using a particular EPR experimental approach. Our approach will allow us to measure interactions in weakly bound protein-protein complex’s even in the presence of a vast excess of unbound labelled protein. Titration based EPR experiments can also accurately determine binding constants.
This project would explore and develop this approach to measurement using novel labelling techniques and EPR experimental methods. Having the ability to measure both affinity and structure will enable us to investigate homologous systems and test hypotheses by sequence engineering.
The project will be supervised jointly between Dundee Lifesciences (Dr Norman) and St Andrews Chemistry (Dr Bode) allowing the student to gain a comprehensive training in protein preparation, derivatisation, EPR techniques and protein structure function relationships. We are equipped with state-of-the-art facilities in both Dundee and St Andrews. We are part of a thriving community of researchers with a broad range of expertise in the application of EPR, both in Dundee and St Andrews.
1) Perkins,J.,Diboun,I.,Dessailly,B.,Lees,J.,Orengo,C. (2010) Transient Protein-Protein Interactions: Structural, Functional, and Network Properties. Structure 18, 1233-1243
2) Vaynberg,J. and Qin,J. (2006) Weak protein–protein interactions as probed by NMR spectroscopy. Trends Biotechnol., 24, 22–27.
3) Cunningham, T., Putterman, M., Desai, A., Horne, W., and Saxena, S. (2015) The Double-Histidine Cu2+-Binding Motif: A Highly Rigid, Site- Specific Spin Probe for Electron Spin Resonance Distance Measurements Angew. Chem. Int.ed. 54, 6330 –6334.