| AVS 63rd International Symposium & Exhibition | |
| Applied Surface Science | Monday Sessions |
| Session AS+BI-MoA |
| Session: | Practical Surface Analysis I: Advancing Biological Surface Analysis/Imaging Beyond ‘Show and Tell’ |
| Presenter: | Elisa Harrison, University of Washington |
| Authors: | E. Harrison, University of Washington G. Interlandi, University of Washington D.G. Castner, University of Washington |
| Correspondent: | Click to Email |
The orientation of adsorbed proteins on surfaces plays a vital role in the function and performance of biomaterials. Development of diagnostic tools such as sandwich ELISAs have focused on controlling the orientation of each protein layer. A full understanding of adsorbed proteins on surfaces, especially at the molecular level, is therefore essential. Our research address es the challenges for characterizing protein orientation by developing new method s to study multilayer protein systems.
The aims of this study were to control and characterize the orientation of protein G B1, an IgG antibody-binding domain of protein G, on well-defined surfaces and measure the effect of its orientation on antibody binding using a variety of surface-sensitive tools and simulations. We hypothesize that binding selectivity would increase for well-ordered protein films due to high er availability of binding domains.
The surface sensitivity of time-of-flight secondary ion mass spectrometry (ToF-SIMS) enables us to distinguish between different proteins and their orientation by monitoring the changes in intensity of amino acid mass fragments. We have developed ToF-SIMS methods for analy zing the orientation of five different cysteine mutants of protein G B1 covalently attached to a maleimide surface. T his technique was further extended by studying multilayer protein systems, specifically the binding of IgG antibodies to the protein G B1 films.
To study the effect of protein orientation on antibody binding, we utilized self-assembled monolayers (SAMs) to form protein G B1 films with both random and well-defined orientations. Using complementary techniques, such as X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation monitoring (QCMD), the ratio of bound IgG antibodies to protein G B1 increased from 0.06, when chemisorbed onto bare gold, to 0.2, when covalent ly attach ed to the surface. Further analysis revealed structure/orientation rearrangement of protein G B1 upon adsorption onto bare gold, which is likely responsible for decreased antibody binding.
Additionally, we developed and applied Monte Carlo (MC) simulations to predict protein orientation on a surface. The MC simulations showed that the outermost b-sheet of protein G B1 interacts most frequently with a hydrophobic surface. The predicted orientations were verified using molecular dynamics simulations, QCMD, and sum frequency generation.
The model systems explored in this study are a first step in developing methodology using state-of-the-art tools that can be applied to more complex systems and expand our knowledge and control of biomolecules on surfaces.