title: Tightening the knot in phytochrome by single-molecule atomic force microscopy.
abstract: A growing number of proteins have been shown to adopt knotted folds. Yet the biological roles and biophysical properties of these knots remain poorly understood. We used protein engineering and atomic force microscopy to explore the single-molecule mechanics of the figure-eight knot in the chromophore-binding domain of the red/far-red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of approximately 47 pN, whereas phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at approximately 73 pN. These forces are not unusual in mechanical protein unfolding, and thus the presence of the knot does not automatically indicate a superstable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting the sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time (to the best of our knowledge), our experiments allow a direct determination of knot size under load. In the unfolded chain, the tightened knot is reduced to 17 amino acids, resulting in apparent shortening of the polypeptide chain by 6.2 nm. Steered molecular-dynamics simulations corroborate this number. Finally, we find that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against a dramatic rearrangement of the native GAF dimer interface upon photoconversion.
authors.affiliation: Department of Physics, Technische Universität München, D-85748 Garching, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bornschlögl
authors.forename: David M
authors.initials: DM
authors.lastname: Anstrom
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mey
authors.forename: Joachim
authors.initials: J
authors.lastname: Dzubiella
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
authors.forename: Katrina T
authors.initials: KT
authors.lastname: Forest
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D034301:Deinococcus; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D010775:Photic Stimulation; D010834:Phytochrome / Q000737:chemistry*; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D055503:Protein Multimerization; D013057:Spectrum Analysis
chemical_list: D010834:Phytochrome