My long term goal is to understand the effects of macromolecular crowding on biochemical processes by acquiring atomic-level information on proteins under actual biological conditions. To this end, my group has pioneered in-cell NMR. We have shown how to assess structure, quantify dynamics, and measure stability under the crowded conditions found in living Escherichia coli cells using this powerful new technique. Now, we want to take the next step. With support from a Pioneer Award, we will focus on using in-cell NMR in eukaryotic cells to study two key proteins in neurodegenerative diseases, the intrinsically disordered proteins, a-synuclein and tau. These proteins are excellent candidates not only because of their disease relevance, but also because we know that macromolecular crowding has extremely large effects on the properties of disordered proteins. Our understanding of protein structure and function has grown enormously in the last 100 years. We have progressed from pondering what role, if any, polypeptides play in the cell, to unraveling, at the atomic level, the mechanisms of enzymes and the molecular bases of protein-protein interactions vital to understanding human disease. Our accumulating wealth of knowledge has largely come from in vitro studies performed under conditions far different from those found in biology. For example, most biochemical examinations of protein behavior are performed at concentrations in the ?g-to-mg-per-mL range, but the insides of cells, where most proteins perform their work, have protein concentrations of >300 mg per mL. Thus, our knowledge comes from data acquired under conditions that are far from physiological relevant, and theory predicts these differences can have extremely large effects on biophysical parameters. Moving beyond the test tube by performing truly in vivo studies in living eukaryotic cells by using NMR spectroscopy is the next frontier in protein chemistry.
| Wang, Yaqiang; Benton, Laura A; Singh, Vishavpreet et al. (2012) Disordered Protein Diffusion under Crowded Conditions. J Phys Chem Lett 3:2703-2706 |
| Zigoneanu, Imola G; Yang, Yoo Jeong; Krois, Alexander S et al. (2012) Interaction of ?-synuclein with vesicles that mimic mitochondrial membranes. Biochim Biophys Acta 1818:512-9 |
| Zigoneanu, Imola G; Pielak, Gary J (2012) Interaction of ýý-synuclein and a cell penetrating fusion peptide with higher eukaryotic cell membranes assessed by ýýýýýF NMR. Mol Pharm 9:1024-9 |
| Barnes, Christopher O; Pielak, Gary J (2011) In-cell protein NMR and protein leakage. Proteins 79:347-51 |
| Fu, Riqiang; Wang, Xingsheng; Li, Conggang et al. (2011) In situ structural characterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR. J Am Chem Soc 133:12370-3 |
| Schlesinger, Alexander P; Wang, Yaqiang; Tadeo, Xavier et al. (2011) Macromolecular crowding fails to fold a globular protein in cells. J Am Chem Soc 133:8082-5 |
| Sharaf, Naima G; Barnes, Christopher O; Charlton, Lisa M et al. (2010) A bioreactor for in-cell protein NMR. J Magn Reson 202:140-6 |
| Li, Conggang; Wang, Gui-Fang; Wang, Yaqiang et al. (2010) Protein (19)F NMR in Escherichia coli. J Am Chem Soc 132:321-7 |
| Wang, Yaqiang; Li, Conggang; Pielak, Gary J (2010) Effects of proteins on protein diffusion. J Am Chem Soc 132:9392-7 |
| Wang, Gui-Fang; Li, Conggang; Pielak, Gary J (2010) Probing the micelle-bound aggregation-prone state of *-synuclein with (19)F NMR spectroscopy. Chembiochem 11:1993-6 |
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