From the Producer Microorganisms to the Lasso Scaffold - Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds - page 25

to 95 °C and in the presence of 6M guanidinium hydrochloride or 8M urea at 65 °C
(Blond et al. 2002 ). Xanthomonins are able to remain unchanged up to 8 h at 95 °C
in water (Hegemann et al. 2014 ). By contrast, other lasso peptides show different
levels of unthreading upon heating. For example caulosegnin I was completely un-
threaded after 4 h at 95 °C and caulosegnin III showed both thermal unthreading and
degradation at the C-terminus (Hegemann et al. 2013a ). Thermal sensibility studies
combined with exoprotease treatments are powerful in determining the amino acids
acting as the plugs by mutagenesis (Hegemann et al. 2014 ).
As mentioned above, MS/MS and MS n may distinguish unambiguously between
the head-to-tail cyclic, branched-cyclic and lasso topologies. Moreover, MS pro-
vides several signatures of the lasso topology upon collision induced dissociation
(CID) or electron capture dissociation (ECD), such as the formation of two-peptide
product ions (upon CID or ECD) (Zirah et al. 2011 ) and H• exchange extent and
differences in dissociation kinetics (upon ECD) (Zirah et al. 2011 ; Pérot-Taillandier
et al. 2012 ). Indeed, in the case of MccJ25, a unique fragmentation behaviour is
observed upon CID, where the C-terminal tail remains trapped non-covalently
within the macrolactam ring forming characteristic two-peptide entities. This fea-
ture allowed assigning rapidly if a MccJ25 variant has acquired or not the lasso
topology (Ducasse et al. 2012b ). However, other lasso peptides do not exhibit such
behaviour. In order to generalize the MS characterization of lasso peptides, other
ionization modes and mass analyses, which allow characterization of the gas-phase
conformation of biomolecules, are required. Therefore, electrospray ionization-
Fourier ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID
or infrared multiple photon dissociation (IRMPD) or electron capture dissociation
(ECD), has been extensively explored and provides a powerful tool for lasso pep-
tide identification (Zirah et al. 2011 ; Pérot-Taillandier et al. 2012 ).

2.3.3 Three-Dimensional Structures

The 3D structures of known lasso peptides are presented in Fig. 2.2 . The topologies
of early discovered lasso peptides were assigned by NMR and molecular modelling
using NOE-derived constraints, such as for RP-71955 (type I) in 1994 (Frechet
et al. 1994 ) and for RES 701-1 (type II) in 1995. Apart from these pioneering
works, very few 3D structures were available in the first years of lasso peptide re-
search (1991-1996). Although MccJ25 led to an impressive number of studies since
1992 on its structure, genetic cluster, mechanism of action and biosynthesis, its 3D
lasso structure was only firmly established by NMR in 2003 to end a long-standing
controversy (Blond et al. 1999 , 2001 ; Bayro et al. 2003 ; Rosengren et al. 2003 ;
Wilson et al. 2003 ; Rebuffat et al. 2004 ).
Since 2008, with the acceleration of lasso peptides discovery and the improve-
ment of high field NMR technology as well as molecular dynamic calculation pro-
grams, an increasing number of lasso topologies have been described in extensive
details (Fig. 2.2 ). The available 3D structures contain one or two short and distorted