Understanding the fundamental molecular mechanisms by which proteins fold into the precise three-dimensional structures required for biological activity remains one of the most challenging problems in structural biology. It is generally accepted that all of the information required for correct folding is coded within the amino acid sequence; just how that code is translated into folding pathways and highly specific tertiary structure is not yet known. There is an urgent need for detailed information on the structures of folding intermediates at the level of individual amino acid residues, and a need for a deeper understanding of the factors that influence their rates of formation and stability. The overall objective of the proposed research is to address some of these outstanding issues through kinetic and equilibrium studies of intermediates formed on the folding pathway of apomyoglobin. Apomyoglobin is ideally suited for such studies because a stable molten globule intermediate that is formed early on the kinetic folding pathway can also be obtained under equilibrium conditions appropriate for high resolution NMR experiments. A combination of mutagenesis, stopped-flow kinetics measurements, hydrogen exchange pulse labeling, and state-of-the-art heteronuclear NMR experiments will be applied to investigate the kinetic folding pathway of apomyoglobin and characterize the structure of the stable molten globule folding intermediate. Mutations will be introduced at specific sites to probe the molecular interactions that stabilize the kinetic folding intermediates, and to investigate the effect of alterations in the intrinsic secondary structural propensities of the polypeptide on the rate of formation and stability of the molten globule intermediate. Direct heteronuclear NMR methods will be used to obtain critical information on the structure, dynamics, and state of hydration of the equilibrium molten globule intermediate of apomyoglobin at the level of single residues. This research program is expected to provide important new insights into the structure and stabilization of folding intermediates at an unprecedented level of detail, and will add significantly to the understanding of protein folding mechanisms. In addition, the kinetic folding pathway of an evolutionarily distant plant leghemoglobin will be characterized, along with the structure of a partly folded intermediate. These studies are expected to provide a particularly stringent test of the hypothesis that folding pathways are conserved in homologous proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK034909-13
Application #
2684143
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1984-12-01
Project End
1999-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
13
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
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; 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
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
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
Meinhold, Derrick W; Wright, Peter E (2011) Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion. Proc Natl Acad Sci U S A 108:9078-83
Nishimura, Chiaki; Dyson, H Jane; Wright, Peter E (2010) Energetic frustration of apomyoglobin folding: role of the B helix. J Mol Biol 396:1319-28
Wright, Peter E; Dyson, H Jane (2009) Linking folding and binding. Curr Opin Struct Biol 19:31-8
Uzawa, Takanori; Nishimura, Chiaki; Akiyama, Shuji et al. (2008) Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR. Proc Natl Acad Sci U S A 105:13859-64

Showing the most recent 10 out of 45 publications