While all the information necessary to encode the secondary and tertiary structure of a protein is contained in its linear sequence of amino acids, translation of this information from primary sequence to native structure often goes awry, resulting in protein mis-folding and aggregation. In some cases, aggregation of proteins can trigger severe cellular dysfunction and disease. Examples include cystic fibrosis, thalassemias, alpha1- antitrypsin deficiency, and several neuropathies such as Alzheimer's, Huntington's and Parkinson's diseases. The progression of these diseases is often correlated with the formation of protein fibrils. However, the growth and deposition of structured fibrils is generally preceded by the formation of amorphous and partially structured, pre-fibrillar states. Growing evidence suggests that pre-fibrillar, low-order aggregates play a central and common role in the pathology of many diseases. Importantly, protein aggregation is heavily influenced by the cellular protein quality control machinery, involving networks of molecular chaperones. Precisely how different chaperone systems cooperate to dismantle and reactivate aggregated proteins, and how molecular chaperone action affects disease progression, is not well understood. A significant impediment to a better understanding of protein aggregate disassembly by molecular chaperones is the inherently complex and heterogeneous nature of an aggregating protein sample. Aggregating proteins typically form a wide variety of conformational states and assemblies. This complex and broad distribution of states is, in general, very difficult to capture with current detection techniques. A principle goal of this proposal is to overcome this analytical limitation in order to develop a detailed mechanistic understanding of how an essential molecular chaperone network, consisting of ClpB, DnaKJ-GrpE and GroEL-ES, extracts and refolds proteins from aggregates. To accomplish this goal, we will: (1) develop a new analytical tool based on single-particle fluorescence burst detection that is capable of rapidly quantifying the specific molar distribution of states within an aggregated protein population, as well as how that distribution changes with time, (2) employ this method to examine the mechanism of protein aggregate disassembly by the DnaKJ-GrpE and ClpB bi-chaperone system, and (3) employ a combination of fluorescence spectroscopy and cryo-electron microscopy to determine how the binding of a non-native protein by DnaK, following extraction from an aggregate, affects the subsequent folding of the protein by GroEL.
Human diseases as diverse as amyotrophic lateral sclerosis, cystic fibrosis, thalassemias, alpha1-antitrypsin deficiency, and a variety of amyloid neuropathies such as Alzheimer's, Huntington's and Parkinson's disease share in common a link to the uncontrolled and pathological self-association of cellular proteins. How this process, known as protein aggregation, leads to disease is not well understood. The progression of protein aggregation inside a living cell is directly influenced by a specialized set of proteins known as molecular chaperones. Our goal is to develop a clear understanding of how protein aggregates are recognized and dismantled by networks of molecular chaperones. A better picture of this process will have a direct impact on our understanding of the large family of diseases that have been linked to protein misfolding and aggregation.
|Duocastella, Martí; Arnold, Craig B; Puchalla, Jason (2017) Selectable light-sheet uniformity using tuned axial scanning. Microsc Res Tech 80:250-259|
|Weaver, Jeremy; Jiang, Mengqiu; Roth, Andrew et al. (2017) GroEL actively stimulates folding of the endogenous substrate protein PepQ. Nat Commun 8:15934|
|Brooks, Arielle; Shoup, Daniel; Kustigian, Lauren et al. (2015) Single particle fluorescence burst analysis of epsin induced membrane fission. PLoS One 10:e0119563|
|Weaver, Jeremy; Watts, Tylan; Li, Pingwei et al. (2014) Structural basis of substrate selectivity of E. coli prolidase. PLoS One 9:e111531|
|Weaver, Jeremy; Rye, Hays S (2014) The C-terminal tails of the bacterial chaperonin GroEL stimulate protein folding by directly altering the conformation of a substrate protein. J Biol Chem 289:23219-32|
|Krantz, Kelly C; Puchalla, Jason; Thapa, Rajan et al. (2013) Clathrin coat disassembly by the yeast Hsc70/Ssa1p and auxilin/Swa2p proteins observed by single-particle burst analysis spectroscopy. J Biol Chem 288:26721-30|
|Chen, Dong-Hua; Madan, Damian; Weaver, Jeremy et al. (2013) Visualizing GroEL/ES in the act of encapsulating a folding protein. Cell 153:1354-65|
|Lin, Zong; Puchalla, Jason; Shoup, Daniel et al. (2013) Repetitive protein unfolding by the trans ring of the GroEL-GroES chaperonin complex stimulates folding. J Biol Chem 288:30944-55|
|Karuri, Nancy W; Lin, Zong; Rye, Hays S et al. (2009) Probing the conformation of the fibronectin III1-2 domain by fluorescence resonance energy transfer. J Biol Chem 284:3445-52|
|Puchalla, Jason; Krantz, Kelly; Austin, Robert et al. (2008) Burst analysis spectroscopy: a versatile single-particle approach for studying distributions of protein aggregates and fluorescent assemblies. Proc Natl Acad Sci U S A 105:14400-5|
Showing the most recent 10 out of 13 publications