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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM066833-10A1
Application #
8610825
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Lewis, Catherine D
Project Start
2003-06-01
Project End
2018-02-28
Budget Start
2014-03-15
Budget End
2015-02-28
Support Year
10
Fiscal Year
2014
Total Cost
$422,323
Indirect Cost
$199,461
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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
Banerjee, Priya R; Moosa, Mahdi Muhammad; Deniz, Ashok A (2016) Two-Dimensional Crowding Uncovers a Hidden Conformation of α-Synuclein. Angew Chem Int Ed Engl 55:12789-92
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:
Deniz, Ashok A (2016) Deciphering Complexity in Molecular Biophysics with Single-Molecule Resolution. J Mol Biol 428:301-7
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
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
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
Banerjee, Priya R; Deniz, Ashok A (2014) Shedding light on protein folding landscapes by single-molecule fluorescence. Chem Soc Rev 43:1172-88
Ferreon, Allan Chris M; Ferreon, Josephine C; Wright, Peter E et al. (2013) Modulation of allostery by protein intrinsic disorder. Nature 498:390-4
Milles, Sigrid; Koehler, Christine; Gambin, Yann et al. (2012) Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range. Mol Biosyst 8:2531-4

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