Research Techniques

Allosteric communication in protein kinases is mediated by structural and dynamic changes of key functional elements such as the activation loop, which regulates both substrate binding and catalysis. These dynamic conformational changes have proven challenging to measure using traditional experimental approaches. The activation loop in particular is highly flexible and is usually invisible in nuclear magnetic resonance experiments (NMR). To solve these problems our group has relied on a range of spectroscopic techniques that have not been widely applied in the kinase field before. This has allowed us to track the activation loop and other regulatory elements of protein kinases and measure how their conformations and dynamics are modulated by protein-protein interactions, post-translational modifications, and drug binding. The results have revealed that protein kinases are far more dynamic than previously recognized and that almost all ligand binding events are coupled to kinase structural changes through long-range allosteric effects. These insights have important implications for kinase activation mechanisms as well as for selective targeting of kinases with inhibitors.

Double electron-electron resonance (DEER) can be used to measure distances in proteins with substantially greater accuracy than FRET. Two paramagnetic spin labels are incorporated by site-specific labeling and the dipolar coupling between the two spins is measured. The technique is capable of providing a distance distribution that can give insight into the populations of different conformational states. This approach has proven ideal for measuring movements of the kinase activation loop between DFG-in (active) and DFG-out (inactive) states.

Time-resolved fluorescence has several advantages over steady-state fluorescence that make it ideal for measuring structural changes in proteins. Most importantly, when performing FRET measurements time-resolve fluorescence allows you to obtain a measure of the distribution of FRET distances in the sample. By engineering FRET sensors that track the activation loop of the kinase we have been able to measure the conformational effects of regulatory interactions and drug binding events in real time and in a high-throughput format amenable to studying many conditions. These experiments have revealed that almost all kinase inhibitors trigger conformational changes upon binding which was not previously appreciated. This allosteric coupling results in drug binding modulating allosteric effectors of the kinase in unexpected ways.   

Nuclear magnetic resonance spectroscopy is a powerful tool for studying protein dynamics. However, its application to protein kinases is complicated by the fact that the key regulatory elements of the kinase like the activation loop undergo movements on the intermediate exchange timescale, resulting in line broadening that can render these important parts of the protein invisible. We have found that this problem can be circumvented by employing paramagnetic relaxation enhancement NMR experiments. A paramagnetic spin label is attached to the activation loop and the location of the spin label is inferred through its effects on other parts of the kinase that are visible in the NMR experiment. 

Infrared spectroscopy is a spectroscopic technique that probes local chemical environment. We use site-directed labeling to incorporate nitrile infrared probes into proteins to interrogate local conformation and dynamics. Cyanophenylalanine nitrile probes can be incorporated co-translationally using amber suppression methodology. The triple bond of the nitrile group absorbs outside the region where the protein itself absorbs rendering it straightforward to detect. We have used this technique to measure the flip of the key DFG motif in Aurora Kinase A for the first time.