Enabling Structural Studies of the Yeast Prion Protein Within a Cellular Environment

My motivation for the work in this thesis was to take steps towards bridging the gap in structural information between atomic models of metastable proteins in isolated and cellular environments. Most biophysical techniques are generally limited by either sample composition or resolution. One technique, Nuclear Magnetic Resonance (NMR) is not limited by sample composition and can provide atomic-level resolution. However, NMR is limited by sensitivity. Recent advancements in the field produced a sensitivity-enhanced solid-state NMR technique, namely Dynamic Nuclear Polarization (DNP) NMR. Using DNP NMR, I observed sensitivity enhancements of up to 90-fold increase in sensitivity, eliminating this barrier. Recent work on a metastable protein, Sup35, assembled in cellular lysates using DNP NMR demonstrates that the biological environment has a dramatic effect on the Sup35 protein structure. In this thesis, I sought to harness sensitivity gains from DNP NMR to identify strategies for the specific detection of isotopically labeled proteins at within a cellular lysate for structural analysis. First I present theoretical calculations, validated by experimental results, for the expected signal of detection ratio of an isotopically labeled protein within a cellular lysate. These results concluded that DNP NMR can specifically detect a 30 kDa, uniformly isotopically labeled protein at low micromolar concentrations. However, sensitivity is still a barrier to specifically detect proteins with lower molecular weights or non-uniform isotopic labeling. Therefore, I optimized sample preparation for maximum sensitivity for DNP NMR on cellular lysates. DNP sensitivity enhancement depends on sample composition. DNP NMR is performed at 100 K, and requires sample glassing, polarizing agent, and protonation for optimal DNP enhancement. Some of the first DNP NMR experiments on purified protein samples were optimized for a matrix of 60:30:10, d8-glycerol:D2O:H2O with 10 mM biradical. These matrix conditions became standard in the field, known as "DNP Juice". However, I found that these matrix conditions are not optimal for DNP NMR cellular lysate samples. In the presence of cellular lysate, sensitivity is improved by addition of lower cryoprotectant (15%) and biradical concentrations (5 mM). I also found that deuteration was unnecessary. Finally, I investigated methods to simplify DNP NMR spectra through segmental labeling of proteins. The strategies in this thesis benefit future research of structural studies on environmentally sensitive proteins, such as alpha-synuclein or tau, within their native environment at physiological concentrations.