(497b) Measuring the Orientation of Electrostatically Immobilized Proteins by Time-of-Flight Secondary Ion Mass Spectrometry and Sum Frequency Generation: From a Model Protein G B1 System to Cytochrome c

The ability to orient proteins on surfaces to control exposure of their biologically active sites will benefit a wide range of applications including protein microarrays, antibody-based diagnostics, affinity chromatography, and biomaterials that present ligands to bind cell receptors. As methods to orient proteins are developed, techniques are required to provide an accurate picture of their orientation. Since no single technique provides a high-resolution image of surface-bound proteins, combinations of surface analytical and spectroscopic techniques are required. In this study, we have developed a model system based on the electrostatic immobilization of a small rigid protein (Protein G B1 domain, 6kDa) to further develop the capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and sum frequency generation (SFG) spectroscopy as tools to probe the orientation of surface immobilized proteins. A Protein G B1 charge mutant (D4) exhibiting net positive and negative charges at either end (for pH 6-8) was produced by neutralizing four negatively charged residues closest to the end of the protein (Aspartic acid to Asparagine or Glutamic acid to Glutamine mutations). These mutants were then immobilized onto amine (NH₃⁺) and carboxyl (COO^-) terminated self assembled monolayers (SAMs) to induce opposite end-on orientations. All SAMs were assembled onto Au via a thiol end group and the quality of these layers were determined by x-ray photoelectron spectroscopy. ToF-SIMS data from the D4 variant on both NH₃⁺ and COO^- SAMs showed intensity differences from secondary ions originating from asymmetric amino acids (Asparagine: 70, 87, and 98 m/z; Methionine: 62 m/z; Tyrosine: 107 and 136 m/z at the N-terminus. Leucine: 86 m/z at the C-terminus). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary ions stemming from residues at either end of the protein. The 50% increase in this ratio, observed between the NH₃⁺ and COO^- SAMs, indicated opposite orientations of the D4 variant on the two different surfaces. In addition, SFG spectral peaks characteristic of ordered alpha-helix (1645 cm^-1) and beta-sheet (1624 and 1675 cm^-1) elements were observed, with a phase that indicated a predominantly upright orientation for the alpha-helix, consistent with an end-on protein orientation. We then moved from this model system and extended this analysis to examine the change in orientation of horse heart Cytochrome c on both NH₃⁺ and COO^- SAMs. The positively charged region at one end of Cytochrome c electrostatically binds to the COO^- substrate while the NH₃⁺ surface elicits the opposite binding orientation. Again, within the SFG spectra, ordering of the protein alpha helices were confirmed by the feature at 1645 cm^-1 and the change in orientation, induced by the two different substrates, is confirmed by intensity differences within ToF-SIMS spectra between ions stemming from asymmetric amino acids (Glutamic acid 84 and 102 m/z; Aspartic acid 72 and 88 m/z).