Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session I
26 – POS
Board 26
Mechanistic Insights into the Folding of Trefoil-knotted Proteins
Nicole Lim
, Sophie E. Jackson.
University of Cambridge, Cambridge, United Kingdom.
The past decade has seen the emergence of a new class of proteins that possess an intriguing
knotted topological feature in their structures formed by the path of the polypeptide backbone.
Elucidating when and how a polypeptide chain knots during the folding represents a significant
challenge to the protein folding field as the knotted topology imposes additional complexity to
the folding landscape. Most of the experimental investigations on knotted proteins have been
focussed on two bacterial trefoil-knotted α/β methyltransferases, YibK and YbeA. Recently, with
the use of a cell-free expression system and pulse-proteolysis kinetic experiments, Mallam and
Jackson were able to investigate the folding rates of nascent chains of knotted proteins after they
were first synthesised by the ribosome.
By using the same cell-free expression system and pulse-proteolysis kinetic experiments, this
study investigates the mechanism of knotting by monitoring the effects of an additional protein
domain on the folding rates of YibK and YbeA when it is fused to either the amino terminus,
carboxy terminus or to both termini. Here, we demonstrate that the fusion of the additional
protein domain to either the carboxy terminus or both termini of the knotted proteins retards the
rate of folding, indicating that the threading motion is hindered. This suggests that the C-
terminus is critical in the threading of the polypeptide chain to form a knot and thus provides the
first experimental evidence of the knotting mechanism. In addition, we also investigate the
effects of the GroEL-GroES chaperonin on the folding of these fusion proteins. Our results shed
light on the role of molecular chaperones on the folding of knotted proteins, thus giving us more
insights as to how knotted proteins have withstood evolutionary pressures despite their complex
topologies and intrinsically slow rates of folding.
27 – POS
Board 27
Untying Peptide Knots: Studies of Lasso Peptide Isopeptidase
A. James Link
.
Princeton University, Princeton, USA.
Lasso peptides are a class of ribosomally-synthesized and posttranslationally-modiifed peptide
(RiPP) that is typified by a structure resembling a slipknot. These peptides consist of 15-25
amino acids, and the slipknot structure is established by the posttranslational installation of an
isopeptide bond between the N-terminus of the peptide and a Glu or Asp sidechain 7-9 amino
acids away. This remarkable structure endows the peptide with protease resistance, making lasso
peptides of interest as scaffolds for peptide drugs. This talk will focus on efforts to discover new
lasso peptides by genome mining. In addition, I will discuss a novel enzyme, lasso peptide
isopeptidase, that specifically cleaves the lasso peptide into a linear form, effectively "untying"
the peptide knot. Hypotheses about the biological role of these lasso peptides and their
isopeptidase will also be discussed.
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