Applications of a Catch and Release Electrospray Ionization Mass Spectrometry Assay for Carbohydrate Library Screening

Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10³ to 10⁶ M^–1 range. Ligands with moderate affinity (10⁴ to 10⁶ M^–1) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (∼10³ M^–1) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ∼10 μM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate–protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein–carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands

Kinetic Stability of the Streptavidin–Biotin Interaction Enhanced in the Gas Phase

Results of the first detailed study of the structure and kinetic stability of the model high-affinity protein–ligand interaction between biotin (B) and the homotetrameric protein complex streptavidin (S₄) in the gas phase are described. Collision cross sections (Ω) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13–139) wild-type (WT) streptavidin, i.e., S₄^<i>n</i>+ and (S₄+4B)^<i>n</i>+ at charge states <i>n</i> = 12–16, were found to be independent of charge state and in agreement (within 10%) with values estimated for crystal structures reported for S₄ and (S₄+4B). These results suggest that significant structural changes do not occur upon transfer of the complexes from solution to the gas phase by electrospray ionization. Temperature-dependent rate constants were measured for the loss of B from the protonated (S₄+4B)^<i>n</i>+ ions. Over the temperature range investigated, the kinetic stability increases with decreasing charge state, from <i>n</i> = 16 to 13, but is indistinguishable for <i>n</i> = 12 and 13. A comparison of the activation energies (<i>E</i>_a) measured for the loss of B from the (S₄+4B)¹³⁺ ions composed of WT streptavidin and five binding site mutants (Trp79Phe, Trp108Phe, Trp120Phe, Ser27Ala, and Tyr43Ala) suggests that at least some of the specific intermolecular interactions are preserved in the gas phase. The results of molecular dynamics simulations performed on WT (S₄+4B)¹²⁺ ions with different charge configurations support this conclusion. The most significant finding of this study is that the gaseous WT (S₄+4B)^<i>n</i>+ ions at <i>n</i> = 12–14, owing to a much larger <i>E</i>_a (by as much as 13 kcal mol^–1) for the loss of B, are dramatically more stable kinetically at 25 °C than the (S₄+4B) complex in aqueous neutral solution. The differences in <i>E</i>_a values measured for the gaseous (S₄+4B)^<i>n</i>+ ions and solvated (S₄+4B) complex can be largely accounted for by a late dissociative transition state and the rehydration of B and the protein binding cavity in solution

Prestress Strengthens the Shell of Norwalk Virus Nanoparticles

We investigated the influence of the protruding domain of Norwalk virus-like particles (NVLP) on its overall structural and mechanical stability. Deletion of the protruding domain yields smooth mutant particles and our AFM nanoindentation measurements show a surprisingly altered indentation response of these particles. Notably, the brittle behavior of the NVLP as compared to the plastic behavior of the mutant reveals that the protruding domain drastically changes the capsid’s material properties. We conclude that the protruding domain introduces prestress, thereby increasing the stiffness of the NVLP and effectively stabilizing the viral nanoparticles. Our results exemplify the variety of methods that nature has explored to improve the mechanical properties of viral capsids, which in turn provides new insights for developing rationally designed, self-assembled nanodevices