Issue 8, 2021

Single molecule force spectroscopy reveals the context dependent folding pathway of the C-terminal fragment of Top7

Abstract

Top7 is a de novo designed protein with atomic level accuracy and shows a folded structure not found in nature. Previous studies showed that the folding of Top7 is not cooperative and involves various folding intermediate states. In addition, various fragments of Top7 were found to fold on their own in isolation. These features displayed by Top7 are distinct from those of naturally occurring proteins of a similar size and suggest a rough folding energy landscape. However, it remains unknown if and how the intra-polypeptide chain interactions among the neighboring sequences of Top7 affect the folding of these Top7 fragments. Here we used single-molecule optical tweezers to investigate the folding–unfolding pathways of full length Top7 as well as its C-terminal fragment (CFr) in different sequence environments. Our results showed that the mechanical folding of Top7 involves an intermediate state that likely involves non-native interactions/structure. More importantly, we found that the folding of CFr is entirely dependent upon its sequence context in which it is located. When in isolation, CFr indeed folds into a cooperative structure showing near-equilibrium unfolding–folding transitions at ∼6.5 pN in OT experiments. However, CFr loses its autonomous cooperative folding ability and displays a folding pathway that is dependent on its interactions with its neighboring sequence/structure. This context-dependent folding dynamics and pathway of CFr are distinct from those of naturally occurring proteins and highlight the critical importance of intra-chain interactions in shaping the overall energy landscape and the folding pathway of Top7. These new insights may have important implications on the de novo design of proteins.

Graphical abstract: Single molecule force spectroscopy reveals the context dependent folding pathway of the C-terminal fragment of Top7

Supplementary files

Article information

Article type
Edge Article
Submitted
19 Nov 2020
Accepted
22 Dec 2020
First published
23 Dec 2020
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2021,12, 2876-2884

Single molecule force spectroscopy reveals the context dependent folding pathway of the C-terminal fragment of Top7

J. Li, G. Chen, Y. Guo, H. Wang and H. Li, Chem. Sci., 2021, 12, 2876 DOI: 10.1039/D0SC06344D

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