Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations and its dynamics

• 成果类型: Article
• 署名作者: Ikeda, Toma; Nojima, Tatsuya; Yamamoto, Souma; Yamada, Ryusei; Niwa, Tatsuya; Konno, Hiroki; Taguchi, Hideki
• 署名单位: Institute of Science Tokyo; Tokyo Institute of Technology; Institute of Science Tokyo; Tokyo Institute of Technology; Kanazawa University; Kanazawa University; Kanazawa University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-15310
• DOI: 10.1073/pnas.2412117122
• 发表日期: 2025-02-18
• 关键词: dihydrofolate-reductase; molecular switch; sequence; principles; variants; binding

摘要

According to classical Anfinsen's dogma, a protein folds into a single unique confor-mation with minimal Gibbs energy under physiological conditions. However, certain proteins may fold into two or more conformations from single amino acid sequences. Here, we designed a protein that adopts interconvertible alternative functional confor-mations, termed seesaw protein (SSP). An SSP was engineered by fusing GFP lacking the C- terminal beta- strand and dihydrofolate reductase (DHFR) lacking the N- terminal beta- strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analyses, including atomic force microscopy (AFM) imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. The drastic conformational change upon the ligand binding was directly visualized by high- speed AFM. Furthermore, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a unique direction for creating artificial proteins with on-off behaviors