Tightening the knot in phytochrome by single molecule atomic force microscopy

TL;DR

This study uses atomic force microscopy and protein engineering to investigate the mechanical properties of a knotted protein domain in phytochrome, revealing unfolding forces, intermediate states, and the size of the tightened knot under load.

Contribution

It provides the first direct measurement of knot size in a protein under load and characterizes the unfolding pathway and stability of phytochrome's knotted domain.

Findings

Apo phytochrome unfolds at ~47 pN, chromophore-bound at ~73 pN.

The tightened knot involves 17 amino acids, shortening the chain by 6.2 nm.

Covalent dimers retain photoreversibility, suggesting stable native interfaces.

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 have used protein engineering and atomic force microscopy to explore single-molecule mechanics of the figure-of-eight knot in the chromophore-binding domain of the red/far red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of ~47 pN, while phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at ~73 pN. These forces are among the lowest measured in mechanical protein unfolding, hence the presence of the knot does not automatically indicate a super-stable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time, our experiments allow 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 found that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against dramatic rearrangement of the native GAF dimer interface upon photoconversion.