The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near edge x-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimideoligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite the cysteine surface position compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0 to 9.5) and salt concentration (0 to 1.5 M NaCl). SFG spectral peaks characteristic of ordered α-helix and β-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the α-helix and β-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s toπ* transition of β-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner β-strands of 40-50° for both variants -one variant more tilted than the other -consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

The surface structure and DNA hybridization performance of thiolated single-strand DNA (HSssDNA) covalently attached to a maleimide-ethylene glycol disulfide (MEG) monolayer on gold have been investigated. Monolayer immobilization chemistry and surface coverage of reactive ssDNA probes were studied by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Orientation of the ssDNA probes was determined by near edge X-ray absorption fine structure (NEXAFS). Target DNA hybridization on the DNA-MEG probe surfaces was measured by surface plasmon resonance (SPR) to demonstrate the utility of these probe surfaces for detection of DNA targets from both purified target DNA samples and complex biological mixtures such as blood serum. Data from complementary techniques showed that immobilized ssDNA density is strongly dependent on the spotted bulk DNA concentration and buffer ionic strength. Variation of the immobilized ssDNA density had a profound influence on the DNA probe orientation at the surface and subsequent target hybridization efficiency. With increasing surface probe density, NEXAFS polarization dependence results (followed by monitoring the N 1s → π* transition) indicate that the immobilized ssDNA molecules reorient towards a more upright position on the MEG monolayer. SPR assays of DNA targets from buffer and serum showed that DNA hybridization efficiency increased with decreasing surface probe density. However, target detection in serum was better on the "high density" probe surface than on the "high efficiency" probe surface. The amount of target detected for both ssDNA surfaces were several orders of magnitude poorer in serum than in purified DNA samples due to non-specific serum protein adsorption onto the sensing surface.

Protein homodimerization is the simplest form of oligomerization that is frequently utilized for the construction of functional biological assemblies and regulation of cellular pathways. Despite its simplicity, dimerization still poses an enormous challenge for protein engineering and chemical maniupulation, owing to the large molecular surfaces involved in this process. We report here the construction of a hybrid coordination motif – consisting of a natural (His) and a non-natural ligand (quinolate) – on the α-helical surface of cytochrome cb562, which a) simultaneously binds divalent metals with high affinity, b) leads to a metal-induced increase in global protein stability, and importantly, c) enables the formation of a discrete protein dimer, whose shape is dictated by the inner-sphere metal coordination geometry. The crystallographically-determined arrangement of metal cross-linked α-helices closely approximate that of the DNA-binding domains of bZIP family transcription factors.

Described is an engineered metal-binding protein, MBPPhen2, which forms porous crystalline frameworks that feature coordinatively unsaturated Zn- and Ni-centers.