Targeting Cellular Lipid Rafts for Dynamic Nuclear Polarization Nuclear Magnetic Resonance.

Lipid rafts serve as important platforms for membrane and signaling proteins. The mechanisms underlying the targeting of nonmyristylated drugs and proteins to lipid rafts remain poorly understood, and the specific structural interactions that govern their localization and stabilization within these membrane microdomains are unclear. This is largely due to a lack of techniques with angstrom resolution that are capable of investigating these membrane microdomains. In-cell nuclear magnetic resonance (NMR) spectroscopy is the only technique with the potential for obtaining such information. Here, we introduce an approach to investigate lipid rafts by in-cell dynamic nuclear polarization (DNP) NMR by covalently linking the polarizing agent AsymPol to the raft-specific protein Ostreolysin A (OlyA). We demonstrate the method's specificity via fluorescence microscopy and obtain DNP enhancements even at very low concentrations of spin-labeled OlyA, whose heterogeneous localization can be identified in DNP buildup curves. Through this work, we identify cross-effect efficiency as a key limiting factor in the pursuit of lipid raft-targeted DNP, revealing important areas of development for enabling targeted DNP of these important cellular structures.