Decoding the information in the primary sequence of a protein is one of the most fundamental challenges in modern biology. A protein's sequence encodes more than just the native structure;it encodes the entire energy landscape - an ensemble of conformations whose energetics and dynamics are finely tuned. The goal of this proposal is a molecular, quantitative, and predictive understanding of the relationship between sequence and the energy landscape together with an understanding of how the environment modulates this landscape. A major hurdle in going from sequence to function is our lack of understanding of the non-native regions of the landscape. High-energy conformations are important for directing the stability and folding of a protein, and modulations of this ensemble play a role in misfolding, protein signaling, catalytic activity, and allostery. While many sequences can encode the same structure, their function and dynamics can vary dramatically. Small variations in a sequence can have effects that range from undetectable to pathological. These differences are often a consequence of subtle changes in the non-native regions of the landscape. Soon we will have access to thousands of human genomes, and without our ability to interpret variation, the potential of these data to impact medicine and human health will never be fully appreciated. It is imperative, therefore, that we have an understanding and control over the relationship between sequence and the energy landscape. Modulations of the energy landscape are not easily detected due to the small populations and transient nature of the high-energy species. The experiments outlined here are aimed at understanding how changes in the sequence and the environment affect the energy landscape.
Aim 1 : Determine how strain influences the energy landscape via single molecule mechanical studies. a. Determine how the geometry of pulling affects the transition state, or barrier for protein folding. b. Determine how the rate-limiting barrier to unfolding changes with force, and how these barriers relate to in vivo mechanical processes such as protein unfoldases.
Aim 2 : Probe the energy landscape through evolution and sequence modulation. a. Use Ancestral Sequence Reconstruction to probe evolutionary changes in the energy landscape. b. Evaluate the role of the hydrophobic core in protein function by selecting for core variants of DNA-binding proteins with altered binding specificities.
Aim 3 : Map unexplored regions of the energy landscape. a. Develop thiol exchange methods to map conformations on the folding reaction coordinate and utilize this kinetic partitioning method to characterize fluctuations on the native side of the transition state. b. Map out the sequence dependence of the earliest events in folding using both rapid mixing techniques and equilibrium models of early folding intermediates.

Public Health Relevance

Decoding the information in a protein's primary sequence is one of the most fundamental challenges in modern biology;this sequence of amino acids specifies the energy landscape, which describes all of a protein's conformations and dynamics and is critical to function. Without our ability to interpret the effects of sequence variation on the energy landscape, the impact of the explosion in human genome sequences will never be realized. The goal of the experiments outlined here is to understand at a molecular, quantitative, and predictive level the relationship between sequence and the energy landscape.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050945-19
Application #
8332755
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1994-05-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
19
Fiscal Year
2012
Total Cost
$332,082
Indirect Cost
$105,858
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Zhuravlev, Pavel I; Hinczewski, Michael; Chakrabarti, Shaon et al. (2016) Reply to Alberti: Are in vitro folding experiments relevant in vivo? Proc Natl Acad Sci U S A 113:E3192
Wheeler, Lucas C; Lim, Shion A; Marqusee, Susan et al. (2016) The thermostability and specificity of ancient proteins. Curr Opin Struct Biol 38:37-43
Lim, Shion A; Hart, Kathryn M; Harms, Michael J et al. (2016) Evolutionary trend toward kinetic stability in the folding trajectory of RNases H. Proc Natl Acad Sci U S A 113:13045-13050
Koulechova, Diana A; Tripp, Katherine W; Horner, Geoffrey et al. (2015) When the Scaffold Cannot Be Ignored: The Role of the Hydrophobic Core in Ligand Binding and Specificity. J Mol Biol 427:3316-26
Bowman, Gregory R; Bolin, Eric R; Hart, Kathryn M et al. (2015) Discovery of multiple hidden allosteric sites by combining Markov state models and experiments. Proc Natl Acad Sci U S A 112:2734-9
Rosen, Laura E; Kathuria, Sagar V; Matthews, C Robert et al. (2015) Non-native structure appears in microseconds during the folding of E. coli RNase H. J Mol Biol 427:443-53
Riedel, Clement; Gabizon, Ronen; Wilson, Christian A M et al. (2015) The heat released during catalytic turnover enhances the diffusion of an enzyme. Nature 517:227-30
Rosen, Laura E; Marqusee, Susan (2015) Autonomously folding protein fragments reveal differences in the energy landscapes of homologous RNases H. PLoS One 10:e0119640
Rosen, Laura E; Connell, Katelyn B; Marqusee, Susan (2014) Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule. Proc Natl Acad Sci U S A 111:14746-51
Hart, Kathryn M; Harms, Michael J; Schmidt, Bryan H et al. (2014) Thermodynamic system drift in protein evolution. PLoS Biol 12:e1001994

Showing the most recent 10 out of 31 publications