The folding of proteins to their native structures is key to their function and malfunction in the cells of living organisms. Protein folding occurs on a high dimensionality and complex surface, whose features are often hidden in standard ensemble studies of protein folding. In this project, we will continue to improve and develop novel single molecule fluorescence methodologies to probe such complex features of protein folding. We will focus on understanding the folding properties of two amyloidogenic proteins, Sup 35 and a-synuclein. The misfolding and aggregation of such amyloidogenic proteins are implicated in a host of diseases including Mad Cow and Parkinson's (a-synuclein). Additionally, mounting evidence recently implicates the aggregation of these proteins in biologically constructive roles, such as acting as a protein-only genetic vehicle in yeast (Sup 35). Hence, a detailed understanding of the folding and dynamics of such proteins is very important from the point of view of human health. We will build on our strong foundation of single molecule investigations of biological folding to further develop and apply a suite of single molecule fluorescence methods, including single molecule FRET, polarization and correlation spectroscopy, in combination with novel and powerful microfluidic methods and protein engineering to gain insights into the folding of these proteins, both as monomeric species, as well as during the early stages of the aggregation process. Our studies will uncover whether these monomeric proteins (both understood to be intrinsically disordered) have elements of residual structure, how repeat sequences influence their folding and dynamics, and how other key cellular factors such as chaperones (for Sup 35) and binding to membranes (for a-synuclein) influence the folding and structural dynamics of such proteins. Furthermore, by monitoring their folding properties during the early stages of aggregation, we aim to understand how these two processes are coupled within the context of this highly complex and heterogeneous mixture of oligomeric species, insight that will be valuable in the understanding of the molecular and structural mechanisms of protein amyloidosis. Finally, these insights are anticipated to be extremely valuable in the design of therapeutic strategies to combat amyloid diseases.

Public Health Relevance

This project aims to develop and apply novel single molecule fluorescence methodologies to probe complex folding features of amyloidogenic proteins. Mechanistic insights gained in these studies will be key in understanding the structural biology of amyloidogenic proteins and protein aggregation, which are implicated in diseases such as Parkinson's and Prion diseases. Insights obtained are expected to be valuable during the design of therapeutic strategies to prevent or reverse such diseases, thus contributing to improving public health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Lewis, Catherine D
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Scripps Research Institute
La Jolla
United States
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Milin, Anthony N; Deniz, Ashok A (2018) Reentrant Phase Transitions and Non-Equilibrium Dynamics in Membraneless Organelles. Biochemistry 57:2470-2477
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
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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