AVS 57th International Symposium & Exhibition
    Biomaterial Interfaces Tuesday Sessions
       Session BI2+AS-TuA

Paper BI2+AS-TuA8
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

Tuesday, October 19, 2010, 4:20 pm, Room Taos A

Session: Combining Techniques for Biointerface Characterization
Presenter: J.E. Baio, University of Washington
Authors: J.E. Baio, University of Washington
T.M. Weidner, University of Washington
L. Baugh, University of Washington
P.S. Stayton, University of Washington
L.J. Gamble, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
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 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 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 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 (Asp to Asn or Glu to Gln mutations). These mutants were then immobilized onto NH3+ and COO- terminated self assembled monolayers (SAMs) to induce opposite end-on orientations. ToF-SIMS data from the D4 variant on both NH3+ and COO- SAMs showed intensity differences from secondary ions originating from asymmetric amino acids (Asn:70, 87, and 98m/z; Met:62m/z; Tyr:107 and 136m/z at the N-terminus. Leu:86m/z at the C-terminus). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of 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 α-helix (1645cm-1) and β-sheet (1624 and 1675cm-1) elements were observed, with a phase that indicated a predominantly upright orientation for the α-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 binds to the COO- substrate while the NH3+ surface elicits the opposite binding orientation. Again, within the SFG spectra, ordering of the protein α-helices were confirmed by the feature at 1645cm-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 (Glu:84 and 102m/z; Asp:72 and 88m/z).