Abstract
Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots. Little is known about their formation or function in proteins despite >1,000 knotted proteins identified in nature. However, these protein knots are not mathematical knots with their backbone polypeptide chains because of their open termini, and the presence of a “knot” depends on the algorithm used to create path closure. Furthermore, it is generally not possible to control the topology of the unfolded states of proteins, therefore making it challenging to characterize functional and physicochemical properties of knotting in any polymer. Covalently linking the amino and carboxyl termini of the deeply trefoil-knotted YibK from Pseudomonas aeruginosa allowed us to create the truly backbone knotted protein by enzymatic peptide ligation. Moreover, we produced and investigated backbone cyclized YibK without any knotted structure. Thus, we could directly probe the effect of the backbone knot and the decrease in conformational entropy on protein folding. The backbone cyclization did not perturb the native structure and its cofactor binding affinity, but it substantially increased the thermal stability and reduced the aggregation propensity. The enhanced stability of a backbone knotted YibK could be mainly originated from an increased ruggedness of its free energy landscape and the destabilization of the denatured state by backbone cyclization with little contribution from a knot structure. Despite the heterogeneity in the side-chain compositions, the chemically unfolded cyclized YibK exhibited several macroscopic physico-chemical attributes that agree with theoretical predictions derived from polymer physics.
Highlights
Knots always fascinate people and have attracted scientists from all disciplines
protein trans-splicing (PTS)-based backbone cyclization relies on the self-association of intein fragments that brings the N- and C-termini together during the protein folding, followed by spontaneous auto-catalytic removal of the intein fragments, thereby achieving backbone cyclization (Scott et al, 1999; Iwai et al, 2001)
The path closure by a backbone peptide bond essentially removes the origin of the protein sequence, rendering obsolete the conventional definition of a protein folding topology by the hierarchical arrangements of secondary structure elements
Summary
Knots always fascinate people and have attracted scientists from all disciplines. Long flexible strings can spontaneously knot themselves upon agitation (Raymer and Smith, 2007). Computational approaches have been used to verify the experimental observations, such as the rugged free energy landscapes of several knotted proteins and multiple intermediates populated along their folding pathways. These computational studies might explain the ratelimiting step of protein knotting (Li et al, 2012; Beccara et al, 2013; Sulkowska and et al, 2013; Faísca, 2015) and lead to various protein knotting mechanisms, such as direct threading, slipknotting, and mousetrapping (Noel et al, 2010; Covino et al, 2014). Proteins are not true mathematical knots but are defined by virtual connections of the N- and C-termini by different mathematical schemes (Taylor, 2000; Lai et al, 2012; Millett et al, 2013)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
