Tightening the knot in phytochrome by single molecule atomic force microscopy

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

2008-09-05

arxiv.org/abs/0809.1067v2

Physics

Biological Physics

12 pages plus five figures; has been submitted to Biophysical J. Replacement on 9/16 is ONLY to correct a typo in the meta dat

Scientific paper

10.1016/j.bpj.2008.11.012

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.

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