Understanding the fundamental molecular mechanisms by which proteins fold remains one of the most challenging problems in structural biology. It is generally accepted that all of the information required for correct folding is contained within the amino acid sequence, but just how that """"""""code"""""""" is translated into folding pathways and the unique three-dimensional structure required for biological activity is not yet known. There is an urgent need for direct experimental information on the structures and dynamics of the numerous conformational states populated in the folding landscape. There is also a need for more detailed understanding of the structure of kinetic folding intermediates and transition state ensembles, and of the fundamental molecular interactions that influence their rates of formation and determine their stability. The overall objective of the proposed research is to address these outstanding issues through kinetic and equilibrium studies of intermediates that populate the folding energy landscape of apomyoglobin. Apomyoglobin provides unique opportunities for detailed investigations of protein folding mechanisms since it exhibits relatively straightforward folding kinetics with well-defined folding intermediates and forms an equilibrium molten globule, similar in structure to the kinetic intermediate, under conditions that allow detailed NMR analysis. In addition, apomyoglobin forms a number of less structured states in acid solution that provide insights into the upper regions of the folding funnel. Multifunctional NMR methods will be used to investigate the structure and dynamics of the pH 4 molten globule, the pH 3 E state, and the pH 2 acid unfolded state of apomyoglobin. These studies will provide insights into the changes in structure and dynamics that accompany chain compaction, at a level of detail that cannot be obtained through kinetic experiments. Mutagenesis coupled with stopped flow kinetics and hydrogen exchange pulse labeling will be used to determine the molecular interactions that stabilize the kinetic molten globule intermediate and the transition state ensemble and influence their structures. This combined NMR and kinetic approach will allow mapping of a protein folding landscape at an unprecedented level of detail.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK034909-20
Application #
6878533
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Sechi, Salvatore
Project Start
1984-12-01
Project End
2006-04-14
Budget Start
2005-04-01
Budget End
2006-04-14
Support Year
20
Fiscal Year
2005
Total Cost
$320,861
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Sun, Xun; Dyson, H Jane; Wright, Peter E (2018) Kinetic analysis of the multistep aggregation pathway of human transthyretin. Proc Natl Acad Sci U S A 115:E6201-E6208
Leach, Benjamin I; Zhang, Xin; Kelly, Jeffery W et al. (2018) NMR Measurements Reveal the Structural Basis of Transthyretin Destabilization by Pathogenic Mutations. Biochemistry 57:4421-4430
Sun, Xun; Jaeger, Marcus; Kelly, Jeffery W et al. (2018) Mispacking of the Phe87 side chain reduces the kinetic stability of human transthyretin. Biochemistry :
Sun, Xun; Dyson, H Jane; Wright, Peter E (2017) Fluorotryptophan Incorporation Modulates the Structure and Stability of Transthyretin in a Site-Specific Manner. Biochemistry 56:5570-5581
Dyson, H Jane; Wright, Peter E (2017) How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway. Acc Chem Res 50:105-111
Lim, Kwang Hun; Dasari, Anvesh K R; Hung, Ivan et al. (2016) Solid-State NMR Studies Reveal Native-like ?-Sheet Structures in Transthyretin Amyloid. Biochemistry 55:5272-8
Aoto, Phillip C; Nishimura, Chiaki; Dyson, H Jane et al. (2014) Probing the non-native H helix translocation in apomyoglobin folding intermediates. Biochemistry 53:3767-80
Li, Xinyi; Zhang, Xin; Ladiwala, Ali Reza A et al. (2013) Mechanisms of transthyretin inhibition of ?-amyloid aggregation in vitro. J Neurosci 33:19423-33
Lim, Kwang Hun; Dyson, H Jane; Kelly, Jeffery W et al. (2013) Localized structural fluctuations promote amyloidogenic conformations in transthyretin. J Mol Biol 425:977-88
Nishimura, Chiaki; Dyson, H Jane; Wright, Peter E (2011) Consequences of stabilizing the natively disordered f helix for the folding pathway of apomyoglobin. J Mol Biol 411:248-63

Showing the most recent 10 out of 50 publications