Method Development for Enhancing Sensitivity of Dynamic Nuclear Polarization Nuclear Magnetic Resonance Spectroscopy for Structural Studies of PKC-Drug Interactions

To perform the most relevant structural studies on biological systems, experiments need to be carried out when the target proteins are in their endogenous cellular environment. Nuclear magnetic resonance (NMR) is well-suited to probe the structure and dynamics of a wide variety of systems, including biologically relevant proteins. However, NMR suffers from an inherent lack of sensitivity. Dynamic nuclear polarization (DNP) NMR is a powerful technique that is used to enhance NMR sensitivity by transferring the greater polarization of exogenously doped electron spins to nuclear spins of interest though the use of a high-power microwave source. Solid effect radicals offer the advantage of being able to be decoupled from nuclear spins with current frequency-agility technology with a technique known as electron decoupling, improving the sensitivity and resolution of DNP NMR. Similarly, frequency-chirped microwaves over the polarization period are shown to enhance the sensitivity of cross effect radicals beyond that which is achievable with conventional continuous wave DNP. Both of these new techniques are shown to be applicable in biologically relevant environments such as intact human cells and cellular lysates. Improving even further upon these sensitivity enhancements will advance the experimental studies of protein kinase c-δ (PKC-δ). Molecular dynamics simulations of the C1b regulatory domain of PKC-δ complexed with various modulators suggest protein-cholesterol interactions play an important role in differentiating the effect of two PKC modulators: phorbol 13-acetate and bryostatin-1. With improved DNP NMR sensitivity, these preliminary in silico results can be tested in vivo