期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：8
DOI：10.1073/pnas.2111231119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
In metallurgy, annealing heats a metal or alloy to a predetermined temperature, holds this temperature for a certain time, and then decreases the metal to room temperature to change the physical and sometimes also the chemical properties of the material. Researchers introduce a similar concept to simulated annealing to predict minimum-energy conformations of biological macromolecules. In this work, we experimentally verify that annealing at a fast cooling rate can synchronize the 70
S ribosome into a nonrotated state with minimum energy in our free-energy landscape analysis. Our results not only offer a facile yet robust approach to stabilizing proteins for high-resolution structural analysis but also contribute to understanding of protein folding and temperature adaptation.
Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70
S ribosome from
Escherichia coli as a model, wherein the 30
S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70
S ribosome homogeneity and improves local resolution on the 30
S subunit. After annealing, the 70
S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70
S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.
