David G Castner oral presentation (OB4-Thu4-1-3)
ToF-SIMS Characterization of Immobilized Protein G Mutants
University of Washington, Box 351653, WA 98195-1653 Seattle, United States
Comprehensive analysis of surface immobilized biomolecules (peptides, proteins, DNA, etc.) with modern surface analysis instrumentation provides an unprecedented level of detail about the immobilization process and the structure of the immobilized biomolecules. ToF-SIMS, when combined with multivariate analysis and other surface analysis methods such as XPS, SFG, NEXAFS, SPR and QCM-D, is a powerful method for obtaining important information about the attachment, type, orientation, conformation and spatial distribution of surface immobilized proteins. Most early ToF-SIMS studies of adsorbed proteins focused on proteins with sizes that were significantly larger than the ToF-SIMS sampling depth (>5nm compared to ~2nm). Recent studies have shown that the orientation can also be determined for Protein G that has a size similar to the ToF-SIMS sampling depth. In this study Protein G B1 variants (6 kDa) with a single cysteine introduced at five different locations in the protein were immobilized in different orientations via the bonding of the cysteine thiol to maleimide oligo(ethylene glycol) (MEG) SAM-covered flat and nanoparticle gold surfaces.
The surface sensitivity of ToF-SIMS was used to distinguish between the different Protein G B1 orientations by monitoring the changes in intensity of characteristic amino acid mass fragments from different locations in the Protein G B1 structure (Ile, Leu and Asn for the C-terminus; Tyr for the N-terminus; Phe and Trp for the middle). The ToF-SIMS (Ile+Leu+Asn)/Tyr was higher for the Protein G B1 mutants with the Cys located near the N-terminus compared to Protein G B1 mutants with the Cys located near the C-terminus for both the MEG covered flat and nanoparticle gold surfaces. This shows that the Protein G B1 orientation can be flipped by changing the location of the Cys from the C-termius to the N- terminus. The opposite orientations of these mutants have implications for the binding interaction between Protein G B1 and immunoglobulin G (IgG) since the binding site for the Fc region of IgG is located near the C-terminus of Protein G B1. QCM-D measurements show that when a monolayer of Protein G is immobilized with the C-terminus facing outward it can bind a monolayer of IgG. Conversely, QCM-D measurements show that when a monolayer of Protein G B1 is immobilized with the N-terminus facing outward it binds very little IgG (~x10 decrease in binding capacity). For the Protein G B1 Cys mutant expected to bind to the MEG SAM in a side-on orientation the ToF-SIMS (Phe+Trp)/Tyr ratio was significantly higher when compared to Protein G B1 mutants that were bound to MEG in end-on orientations.