Tightening the knot in phytochrome by single-molecule atomic force microscopy

Area of Science:

• Biophysics
• Protein Folding
• Molecular Biology

Background:

• Proteins with knotted folds are increasingly identified, but their biological functions and mechanical properties are not well understood.
• Phytochrome, a red/far-red photoreceptor, contains a chromophore-binding domain with a figure-eight knot.

Purpose of the Study:

• To investigate the single-molecule mechanics of the figure-eight knot in phytochrome using protein engineering and atomic force microscopy.
• To determine the mechanical unfolding forces and characterize the unfolding pathway of knotted phytochrome.
• To measure the size of the knot under mechanical load and assess its impact on protein stability.

Main Methods:

• Single-molecule atomic force microscopy (AFM) was employed to mechanically unfold phytochrome.
• Protein engineering techniques were used to create and manipulate phytochrome constructs.
• Steered molecular dynamics (MD) simulations were performed to corroborate experimental findings.

Main Results:

• Apo phytochrome unfolded at ~47 pN, while chromophore-bound phytochrome unfolded at ~73 pN, indicating the knot itself doesn't confer super-stability.
• A stable intermediate was observed during mechanical unfolding, suggesting sequential unfolding of subdomains (potentially GAF and PAS domains).
• The figure-eight knot was directly measured under load, reducing to 17 amino acids and shortening the chain by 6.2 nm, confirmed by MD simulations.

Conclusions:

• The figure-eight knot in phytochrome does not significantly increase unfolding resistance, challenging assumptions about knot-induced protein super-stability.
• Mechanical unfolding reveals distinct subdomains within phytochrome and provides the first direct measurement of knot size under load.
• Phytochrome dimers retain photoreversibility, suggesting limited rearrangement of the GAF dimer interface during photoconversion.