RTA1-33/44-198 is a catalytically inactive, single-domain derivative of the @entity70266 ( @entity70266 ) engineered to serve as a stable protein scaffold for presentation of native immunogenic epitopes (Olson et al., Protein Eng Des Sel 2004;17:391-397). To improve the stability and solubility of RTA1-33/44-198 further, we have undertaken the design challenge of introducing a @entity1520 (SS) bond. Nine pairs of residues were selected for placement of the SS-bond based on molecular dynamics simulation studies of the modeled single-domain chain. @entity1520 formation at either of two positions ( @entity70267 / @entity49730 or @entity70268 /E99C) involving a specific surface loop (44-55) increased the protein melting temperature by ~5  C compared with RTA1-33/44-198 and by ~13  C compared with @entity70266 . Prolonged stability studies of the @entity70267 / @entity49730 variant (> 60 days at 37  C, pH 7.4) confirmed a > 40% reduction in self-aggregation compared with RTA1-33/44-198 lacking the SS-bond. The @entity70267 / @entity49730 variant retained affinity for anti- @entity70266 antibodies capable of neutralizing ricin toxin, including a monoclonal that recognizes a @entity1 B-cell epitope. Introduction of either @entity70267 / @entity49730 or @entity70268 /E99C promoted crystallization of RTA1-33/44-198, and the X-ray structures of the variants were solved to 2.3 A or 2.1 A resolution, respectively. The structures confirm formation of an intramolecular SS-bond, and reveal a single-domain fold that is significantly reduced in volume compared with @entity70266 . Loop 44 to 55 is partly disordered as predicted by simulations, and is positioned to form self-self interactions between symmetry-related molecules. We discuss the importance of @entity70266 loop 34 to 55 as a nucleus for unfolding and aggregation, and draw conclusions for ongoing structure-based minimalist design of @entity70266 -based immunogens.

Introduction of a XXXX bond leads to stabilization and crystallization of a ricin immunogen.

@entity1 :: ('9606', 'Species') :: ['human'] @entity70266 :: ('23543', 'Gene') :: ['RTA', 'ricin toxin A-chain'] @entity70267 :: ('p|SUB|R|48|C', 'ProteinMutation') :: ['R48C'] @entity49730 :: ('c|SUB|T|77|C', 'DNAMutation') :: ['T77C'] @entity1520 :: ('CHEBI:48343', 'Chemical') :: ['Disulfide', 'disulfide'] @entity70268 :: ('p|SUB|V|49|C', 'ProteinMutation') :: ['V49C']
@entity1520 :: (CHEBI:48343,Chemical) :: ['disulfide']
