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Sequence-encoded folding is the foundation of protein structure and is also possible in synthetic chains of artificial chemical composition. In natural proteins, the characteristics of the unfolded state are as important as those of the folded state in determining folding energetics. While much is known about folded structures adopted by artificial protein-like chains, corresponding information about the unfolded states of these molecules is lacking. Here, we report the consequences of altered backbone composition on the structure, stability, and dynamics of the folded and unfolded states of a compact helix-rich protein. Characterization through a combination of biophysical experiments and atomistic simulation reveals effects of backbone modification that depend on both the type of artificial monomers employed and where they are applied in sequence. In general, introducing artificial connectivity in a way that reinforces characteristics of the unfolded state ensemble of the prototype natural protein minimizes the impact of chemical changes on folded stability. These findings have implications in the design of protein mimetics and provide an atomically detailed picture of the unfolded state of a natural protein and artificial analogues under non-denaturing conditions.
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Analysis of folded structure and folding thermodynamics in heterogeneous-backbone proteomimetics
Recent years have seen a growing number of examples of designed oligomeric molecules with artificial backbone connectivity that are capable of adopting complex folded tertiary structures analogous to those seen in natural proteins. A range of experimental techniques from structural biology and biophysics have been brought to bear in the study of these proteomimetic agents. Here, we discuss some considerations encountered in the characterization of high-resolution folded structure as well as folding thermodynamics of protein-like artificial backbones. We provide an overview of the use of X-ray crystallography and NMR spectroscopy in such systems and review example applications of these methods in the primary literature. Further, we provide detailed protocols for two experiments that have proved useful in our prior and ongoing efforts to compare folding thermodynamics between natural protein domains and heterogeneous-backbone counterparts.
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- Award ID(s):
- 1807301
- PAR ID:
- 10293681
- Editor(s):
- Petersson, E. James
- Date Published:
- Journal Name:
- Methods in enzymology
- Volume:
- 656
- ISSN:
- 0076-6879
- Page Range / eLocation ID:
- 93-122
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/bs.mie.2021.04.009
