  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.
