Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation

doi:10.1073/pnas.0601567103

Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation

Departments of ^†Physics,
^¶Biochemistry and Molecular Biology, and
^††Chemistry and
^‡Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637

Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved June 12, 2006 (received for review February 24, 2006)

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 ≈3 nm (at the 10-pN detection limit of the measurement) and destabilized by ≈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.

Footnotes

^‡‡To whom correspondence should be sent at the present address:
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078.
E-mail: wouter.hoff{at}okstate.edu
↵ ^§Present address: Department of Radiology, Johns Hopkins University, Baltimore, MD 21205.
↵ ^‖Present address: Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208.
Author contributions: J.M.Z., N.F.S., and W.D.H. designed research; J.M.Z., H.L., and R.A.N. performed research; H.L. and S.M. contributed new reagents/analytic tools; J.M.Z., R.A.N., and W.D.H. analyzed data; and J.M.Z., N.F.S., and W.D.H. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

PAS,

Per-Arnt-Sim;

PYP,

photoactive yellow protein;

MD,

molecular dynamics;

SMD,

steered MD.