Identification of an on- pathway intermediate illuminates the kinetic competition between protein folding and misfolding

• 成果类型: Article
• 署名作者: Luan, Qing; Clark, Patricia L.
• 署名单位: University of Notre Dame
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14260
• DOI: 10.1073/pnas.2425999122
• 发表日期: 2025-08-05
• 关键词: autotransporter virulence protein; contact order; mechanism; aggregation; secretion; EVOLUTION; SEQUENCES; transport; reveals; im7

摘要

Our current understanding of protein folding is based predominantly on studies of small (<150 aa) proteins that refold reversibly from a chemically denatured state. However, as protein length increases so does the competition between off-pathway misfolding and on-pathway folding, creating a more complex energy landscape (folding funnel). Little is known about how intermediates populated during the folding of larger proteins affect navigation of this more complex landscape. Previously, we reported extremely slow folding rates for the 539 aa beta- helical passenger domain of pertactin (P.69T), including conditions that favor the formation of a kinetically trapped, off-pathway partially folded state (PFS). Existence of an on-pathway intermediate for P.69T folding was speculated but its characterization remained elusive. In this work, we exploited the extremely slow kinetics of PFS unfolding to develop a double-jump denaturant challenge assay. With this assay, we identified a transient unfolding intermediate, PFS*, that adopts a similar structure to PFS, including C-terminal folded structure and a disordered N terminus, yet unfolds much more quickly than PFS. Additional experiments revealed that PFS* also functions as an on-pathway intermediate for P.69T folding. Collectively, these results support a C-to-N-terminal model for P.69T folding, with folding initiated in the C-terminus with the rate-limiting formation of the transient on-pathway PFS* intermediate, which sits at the junction of the kinetic competition between folding and misfolding. Notably, processive folding from C-to-N terminus also occurs during C-to-N-terminal translocation of P.69T across the bacterial outer membrane. These results illuminate the crucial role of kinetics when navigating a complex energy landscape for protein folding.