The long-term goal of this project is to understand beta-sheet folding energetics at a level that enables protein and proteomimetic design for therapeutic purposes. Comprehending beta-sheet folding energetics and contrasting them with misfolding energetics is essential for understanding the role of protein folding in health and disease, and for designing backbone modified proteins with enhanced physical properties.
In specific aim 1 (50% effort), we employ a backbone and side chain mutagenesis strategy to test the hypothesis that a few energetically key residues make context-dependent, synergistic, hydrophobic and H-bonding contributions to native state stability. Such residues may be identifiable a priori owing to their extent of solvent exposure. Furthermore, we test the idea that backbone amide transfer free energies may be sensitive to the size and type of flanking side chains;if so this feature can easily be factored into beta-sheet design. In addition, we will explore the possibility of replacing pairs of solvent shielded backbone amides that make stabilizing H-bonds with E-olefin dipeptide isosteres to discern whether a backbone- backbone hydrophobic interaction could replace one or more H-bonds. This is the first step towards designing and synthesizing proteomimetics that fold and function despite having significantly fewer amide bonds than their ribosomally derived counterparts. Such mimetics could have substantially better membrane permeability properties than standard proteins, perhaps enabling aerosol and oral bioavailability.
In specific aim 2 (50% effort), we take on the challenging task of understanding how N-glycosylation influences beta-sheet folding energetics, so that we can integrate this information into protein folding and secretion models that also consider the role of chaperones, folding enzymes, and degradation machinery. Any factor that influences protein folding kinetics and thermodynamics is likely to affect the partitioning of proteins between degradation pathways and normal folding and trafficking pathways. We test several hypotheses, including the idea that N-glycosylation of asparagine residues influences beta-sheet folding kinetics and thermodynamics, that a phi-value analysis as a function of oligosaccharide structure will reveal molecular details of the mechanism by which N-glycosylation influences folding, and that multiple glycosylation sites collaborate to influence folding of immunoglobulin domains, the fold found in antibody drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051105-15
Application #
7907599
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Wehrle, Janna P
Project Start
1994-09-01
Project End
2012-04-30
Budget Start
2010-06-01
Budget End
2012-04-30
Support Year
15
Fiscal Year
2010
Total Cost
$358,114
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Ardejani, Maziar S; Powers, Evan T; Kelly, Jeffery W (2017) Using Cooperatively Folded Peptides To Measure Interaction Energies and Conformational Propensities. Acc Chem Res 50:1875-1882
Chen, Wentao; Kong, Leopold; Connelly, Stephen et al. (2016) Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon. ACS Chem Biol 11:1852-61
Dave, Kapil; J├Ąger, Marcus; Nguyen, Houbi et al. (2016) High-Resolution Mapping of the Folding Transition State of a WW Domain. J Mol Biol 428:1617-36
Hsu, Che-Hsiung; Park, Sangho; Mortenson, David E et al. (2016) The Dependence of Carbohydrate-Aromatic Interaction Strengths on the Structure of the Carbohydrate. J Am Chem Soc 138:7636-48
Chen, Wentao; Dong, Jiajia; Li, Suhua et al. (2016) Synthesis of Sulfotyrosine-Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy. Angew Chem Int Ed Engl 55:1835-8
Murray, Amber N; Chen, Wentao; Antonopoulos, Aristotelis et al. (2015) Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity. Chem Biol 22:1052-62
Hebert, Daniel N; Lamriben, Lydia; Powers, Evan T et al. (2014) The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis. Nat Chem Biol 10:902-10
Chen, Wentao; Enck, Sebastian; Price, Joshua L et al. (2013) Structural and energetic basis of carbohydrate-aromatic packing interactions in proteins. J Am Chem Soc 135:9877-84
Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim et al. (2012) Correction to Context-Dependent Effects of Asparagine Glycosylation on Pin WW Folding Kinetics and Thermodynamics. J Am Chem Soc 134:4450-4451
Araya, Carlos L; Fowler, Douglas M; Chen, Wentao et al. (2012) A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc Natl Acad Sci U S A 109:16858-63

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