The goal of this proposal is to understand how the amino acid sequence of a protein dictates its fold and stability. In order to accomplish this goal, we need to understand the structure and stability of all the conformations accessible to a given sequence, i.e. its energy landscape. The difficult with this is that proteins are extremely cooperative. In order to bypass this problem, we have dissected a protein's structure and stability into smaller pieces and studied both the partially-folded conformations and folded parts of the protein E. coli ribonuclease H. by comparing the results from kinetic and equilibrium experiments, we have obtained evidence for a surprising conclusion: all of the partially folded conformations (the acid molten globule, the higher energy conformations of the native state and a kinetic folding intermediate) are structured in the same specific regions of the protein. RNase H folds in an apparently hierarchical fashion in which the most stable individual element folds first. These results suggest important implications about the relationship between protein stability, structure and folding. The experiments outlined in this proposal explore the implications of this work to learn what makes one region of the protein the most stable and how the overall energy landscape of a protein affects its global stability, folding and dynamics. Specifically, the aims of this proposal are: 1. Explore the energy landscape of E. coli RNase H using site-specific mutagenesis, testing the relationship between the hierarchy of stability in the native state and the folding pathways(s) of the protein. 2. Determine the structure, stability and folding of autonomous folding fragments comprising the folding core of RNase H. 3. Determine the energy landscape of a thermophilic RNase H (T. thermophilus). The feature of the structure, stability and folding of this protein will be compare to the mesophilic homologue to see if the general rules for thermostability lie in the distribution of energy within the protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM050945-06
Application #
2852381
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1994-05-01
Project End
2003-05-31
Budget Start
1999-06-01
Budget End
2000-05-31
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Samelson, Avi J; Bolin, Eric; Costello, Shawn M et al. (2018) Kinetic and structural comparison of a protein's cotranslational folding and refolding pathways. Sci Adv 4:eaas9098
Zhang, Yongli; Ha, Taekjip; Marqusee, Susan (2018) Editorial Overview: Single-Molecule Approaches up to Difficult Challenges in Folding and Dynamics. J Mol Biol 430:405-408
Guinn, Emily J; Tian, Pengfei; Shin, Mia et al. (2018) A small single-domain protein folds through the same pathway on and off the ribosome. Proc Natl Acad Sci U S A 115:12206-12211
Guinn, Emily J; Marqusee, Susan (2018) Exploring the Denatured State Ensemble by Single-Molecule Chemo-Mechanical Unfolding: The Effect of Force, Temperature, and Urea. J Mol Biol 430:450-464
Lim, Shion An; Bolin, Eric Richard; Marqusee, Susan (2018) Tracing a protein's folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange. Elife 7:
Lim, Shion A; Marqusee, Susan (2018) The burst-phase folding intermediate of ribonuclease H changes conformation over evolutionary history. Biopolymers 109:e23086
Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K et al. (2017) An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer. J Biol Chem 292:15636-15648
Samelson, Avi J; Jensen, Madeleine K; Soto, Randy A et al. (2016) Quantitative determination of ribosome nascent chain stability. Proc Natl Acad Sci U S A 113:13402-13407
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

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