title: Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation.
abstract: The Per-Arnt-Sim (PAS) domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. The activation of these functions is thought to involve partial unfolding of N- or C-terminal helices attached to the PAS domain. Here we use atomic force microscopy to probe receptor activation in single molecules of photoactive yellow protein (PYP), a prototype of the PAS domain family. Mechanical unfolding of Cys-linked PYP multimers in the presence and absence of illumination reveals that, in contrast to previous studies, the PAS domain itself is extended by approximately 3 nm (at the 10-pN detection limit of the measurement) and destabilized by approximately 30% in the light-activated state of PYP. Comparative measurements and steered molecular dynamics simulations of two double-Cys PYP mutants that probe different regions of the PAS domain quantify the anisotropy in stability and changes in local structure, thereby demonstrating the partial unfolding of their PAS domain upon activation. These results establish a generally applicable single-molecule approach for mapping functional conformational changes to selected regions of a protein. In addition, the results have profound implications for the molecular mechanism of PAS domain activation and indicate that stimulus-induced partial protein unfolding can be used as a signaling mechanism.
authors.affiliation: Department of Physics, University of Chicago, IL 60637, USA.
authors.forename: Jason Ming
authors.initials: JM
authors.lastname: Zhao
authors.forename: Haeshin
authors.initials: H
authors.lastname: Lee
authors.forename: Rene A
authors.initials: RA
authors.lastname: Nome
authors.forename: Sophia
authors.initials: S
authors.lastname: Majid
authors.forename: Norbert F
authors.initials: NF
authors.lastname: Scherer
authors.forename: Wouter D
authors.initials: WD
authors.lastname: Hoff
mesh_terms: D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D006801:Humans; D008027:Light; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria