Why certain proteins are unable to express the spatial information encoded in their amino acid sequences without the aid of molecular chaperones is not fully understood. Yet protein misfolding underlies devastating pathologies, such as cystic fibrosis, thalassemias, and a variety of amyloid neuropathies such as Alzheimer's and Huntington's disease. The long-term goal of this proposal is to understand how molecular chaperones guide proteins to their final, active three dimensional structures. Toward this end, we intend to focus on one subclass of the molecular chaperones, the ubiquitous, ring-shaped complexes known as chaperonins. One member of this conserved and essential family of proteins is the bacterial GroEL-GroES complex. Using the GroEL-GroES system as a model, along with model folding substrates, we propose experiments intended to uncover general principles for chaperone-dependent protein folding. In order to study the dynamics of the GroEL chaperonin and how it interacts with protein folding intermediates, we are developing fluorescence and rapid mixing methods. Previously, we successfully used fluorescence energy transfer (FRET) to follow the sequence of steps that drive the GroEL reaction cycle. This method relies on the introduction of cysteine residues into GroEL, GroES and substrate protein, which are then labeled with fluorescent probes. We now extend this approach to include time-resolved FRET measurements, in order to systematically map the morphology of a GroEL-bound folding intermediate. We anticipate that this combined approach will allow us to determine why the GroEL-GroES chaperonin is required to fold certain proteins and how specific interactions between these proteins, GroEL, and GroES facilitate productive folding.
Our aims are: (1) to develop a FRET assay which can be used to map the morphology of a GroEL bound folding intermediate, (2) to apply this assay to follow structural changes in a folding intermediate while bound to GroEL in order to test two models of GroEL-stimulated folding and (3) to determine how GroEL-dependent protein folding is triggered, by testing specific models of substrate encapsulation beneath GroES.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM065421-01A1
Application #
6577369
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
1
Fiscal Year
2003
Total Cost
$276,022
Indirect Cost
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
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

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