The fascinating process by which proteins fold to complex 3-dimensional structures plays critical roles in their myriad functions in cells and organisms, and in misfolding that can lead to disease. Protein misfolding and aggregation (amyloid formation) are implicated in several diseases such as Parkinson's and Alzheimer's diseases, prion diseases, diabetes, and heart disease. Furthermore, recent work supports the intriguing idea that protein amyloids can also have functional roles in biology. Therefore, understanding the mechanisms by which proteins fold is critical to furthering our understanding of basic biology and disease, understanding that could later assist with design of therapeutic strategies in this regard. While much has been learned about protein folding and aggregation, these are extremely complex processes, leaving a host of issues yet to be resolved. One aspect of recent note along these lines is the increasing realization that a spectrum of protein disorder (flexibility) is prevalent in genomes, and that this disorder is often intimately linked to functio and malfunction. For example, monomeric forms of several amyloid-forming proteins have extensive stretches of disordered sequence. Here, we propose to develop and apply novel single-molecule fluorescence methods to take next key steps in furthering our understanding of several of the above aspects. Our work will focus on the Parkinson's disease-linked protein ?-synuclein, which has multiple putative biological functions. This protein has substantial disorder as a monomer and can fold upon binding to partners, an interesting feature that it shares with many disordered proteins. Although ?-synuclein has been investigated for many years, limited insight has been gained about its complex and dynamic biophysics. By avoiding the averaging inherent in most ensemble experiments, we will probe the complex folding landscape of this system and its natural variants during interactions and aggregation at a resolution not feasible by standard methods. Our innovative work will uniquely combine facets of cutting-edge biophysics, optics, biochemistry, and chemical biology. Overall, the work will result in a new level of understanding of the ?-synuclein system, with implications for better understanding of the biology of neurodegenerative diseases. Furthermore, we will develop and implement combination, state-of-the-art single-molecule tools which will be broadly applicable in studies of a host of other biologically important molecules.

Public Health Relevance

We will study the complex biophysics of the Parkinson's disease-related protein a-synuclein, including its folding, interaction with partners and aggregation. Insights gained are expected to be valuable for the future design of therapeutic strategies to prevent or reverse such protein misfolding diseases, thus contributing to improving public health. The work will also result in new state-of-the-art tools broadly applicable to a number of biological systems.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Lewis, Catherine D
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Scripps Research Institute
La Jolla
United States
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Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A et al. (2018) Site-Specific Three-Color Labeling of ?-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation. Cell Chem Biol 25:797-801.e4
Milin, Anthony N; Deniz, Ashok A (2018) Reentrant Phase Transitions and Non-Equilibrium Dynamics in Membraneless Organelles. Biochemistry 57:2470-2477
Banerjee, Priya R; Milin, Anthony N; Moosa, Mahdi Muhammad et al. (2017) Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets. Angew Chem Int Ed Engl 56:11354-11359
Lee, Taehyung C; Kang, Minjin; Kim, Chan Hyuk et al. (2016) Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding. Chembiochem 17:981-4
Deniz, Ashok A (2016) Deciphering Complexity in Molecular Biophysics with Single-Molecule Resolution. J Mol Biol 428:301-307
Banerjee, Priya R; Moosa, Mahdi Muhammad; Deniz, Ashok A (2016) Two-Dimensional Crowding Uncovers a Hidden Conformation of ?-Synuclein. Angew Chem Int Ed Engl 55:12789-12792
Banerjee, Priya R; Mitrea, Diana M; Kriwacki, Richard W et al. (2016) Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding. Angew Chem Int Ed Engl 55:1675-9
Mitrea, Diana M; Cika, Jaclyn A; Guy, Clifford S et al. (2016) Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. Elife 5:
Moosa, Mahdi Muhammad; Ferreon, Allan Chris M; Deniz, Ashok A (2015) Forced folding of a disordered protein accesses an alternative folding landscape. Chemphyschem 16:90-4
Lee, Taehyung; Moran-Gutierrez, Crystal R; Deniz, Ashok A (2015) Probing protein disorder and complexity at single-molecule resolution. Semin Cell Dev Biol 37:26-34

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