13 research outputs found
Tightening the knot in phytochrome by single molecule atomic force microscopy
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.Comment: 12 pages plus five figures; has been submitted to Biophysical J.
Replacement on 9/16 is ONLY to correct a typo in the meta data; the uploaded
file is identical to first versio
Knotted vs. Unknotted Proteins: Evidence of Knot-Promoting Loops
Knotted proteins, because of their ability to fold reversibly in the same topologically entangled conformation, are the object of an increasing number of experimental and theoretical studies. The aim of the present investigation is to assess, on the basis of presently available structural data, the extent to which knotted proteins are isolated instances in sequence or structure space, and to use comparative schemes to understand whether specific protein segments can be associated to the occurrence of a knot in the native state. A significant sequence homology is found among a sizeable group of knotted and unknotted proteins. In this family, knotted members occupy a primary sub-branch of the phylogenetic tree and differ from unknotted ones only by additional loop segments. These "knot-promoting" loops, whose virtual bridging eliminates the knot, are found in various types of knotted proteins. Valuable insight into how knots form, or are encoded, in proteins could be obtained by targeting these regions in future computational studies or excision experiments
Entangled Proteins: Knots, Slipknots, Links, and Lassos
In recent years the studies of entangled proteins have grown into the whole new, interdisciplinary and rapidly developing field of research. Here we present various types of entangled proteins studied within this field, which form knots, slipknots, links, and lassos. We discuss their geometric features and indicate what biological and physical role the entanglement plays. We also discuss mathematical tools necessary to analyze such structures and present databases and servers assembling information about entangled proteins: KnotProt, LinkProt, and LassoProt