Paper BI-TuM9
Surface Immobilization and Characterization of Proteins

Tuesday, October 21, 2008, 10:40 am, Room 202

Immobilized proteins mediate the interactions between a material and its biological environment. We have used XPS, ToF-SIMS, NEXAFS and SPR to investigate protein immobilization onto surfaces containing nitrilotriacetic acid (NTA), N-hydroxysuccinimide (NHS) and maleimide headgroups. NHS surfaces were prepared by self-assembly of NHS ester oligo(ethylene glycol) thiols (NHS-OEG) onto gold. Protein immobilization onto NHS surfaces occurs primarily through the amine groups on the side chains of lysine residues present on the protein surface, resulting in the proteins being immobilized in a random orientation. Mixed monolayers containing NTA headgroups and OEG chains were self-assembled onto a gold surface. The surface concentration of NTA headgroups was 0.9-1.3 molecule/nm² in the mixed NTA/OEG monolayers, compared to 1.9 molecule/nm² in pure NTA monolayers. The NTA headgroups were slightly reoriented toward an upright position after OEG incorporation. Histagged, proteins were specifically and reversibly immobilized onto Ni(II)-treated mixed NTA monolayers in well-defined orientations. For a humanized anti-lysozyme Fv fragment the amount of reversible, site-specific adsorption varied from 108 - 205 ng/cm² with dissociation rates (k_off) between 1x10^-4 and 2x10^-5 s^-1, both depending on the NTA surface concentration and orientation. The monolayers without Ni(II) treatment exhibited low nonspecific adsorption. ToF-SIMS was used to compare the controlled orientation of histagged proteins on NTA surfaces with the random orientation of proteins on NHS surfaces. Previously studies have characterized the composition and structure of maleimide-ethylene glycol disulfide (MEG) monolayers on gold for the immobilization of single-stranded DNA oligomers (Lee, et al., Analytical Chemistry 79 (2007) 4390.). These same MEG surfaces were used to covalently immobilize cysteine mutants of the Protein G B1 domain. Two mutants were prepared with cysteines located at opposite ends of the Protein G B1 domain. XPS and SPR were used to quantify the amount of each cysteine mutant onto both bare gold and MEG covered gold surfaces. The ToF-SIMS intensity ratios of amino acid fragments with asymmetric distributions in the Protein G B1 domain (ala, asn, gly, leu, met and tyr) were used to show the two immobilized cysteine mutants had opposite orientations. This difference in orientation was observed on both the gold and MEG surfaces.