Orientation of Biotin-Binding Sites in Streptavidin Adsorbed onto the Surface of Polythiophene Films

Abstract

The orientation of biotin-binding sites of streptavidin adsorbed to thin films of three polythiophenes (PTs), namely, regioregular poly(3-hexylthiophene) (RP3HT), regiorandom poly(3-butylthiophene) (P3BT), and poly(3,3‴-didodecylquaterthiophene) (PQT12), has been investigated. Polymer films were examined prior to and after protein adsorption with atomic force microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA) applied to ToF-SIMS data revealed subtle changes in surface chemistry of polymer films and orientation of adsorbed streptavidin. PCA resolved the surface alignment of alkyl side chains and differentiated the ToF-SIMS data for PQT12, RP3HT, and P3BT, verifying an amorphous morphology for P3BT and a semicrystalline one for PQT12 and RP3HT. After the characterization of the polymeric films, streptavidin adsorption from solutions with different protein concentrations (up to 300 μg/mL) has been conducted. The PCA results distinguished between amino acids characteristic for external regions of streptavidin molecules adsorbed to different PTs suggest that streptavidin adsorbed to PQT12 exposes molecular regions rich in tryptophan and tyrosine, which are components of the biotin-binding sites. The latter results were confirmed using biotin-labeled horse radish peroxidase to estimate the exposed binding sites of streptavidin adsorbed onto the different PT films. The analysis of streptavidin structure suggests that interaction between polythiophene film and dipole moment of streptavidin subunit is responsible for orientation of biotin-binding sites.

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

“…(67,68) Although each monomer of wild-type streptavidin has a calculated dipole moment of 256 D according to the Weizmann server, (69,70) the individual moments of the monomers cancel each other out when aligned in the tetramer. (19,40,71) Such cancellation of the invidiual moments of the monomers does not occur for monovalent streptavidin. Slight variations in the structure of MSA compared to that of wild-type streptavidin introduce a dipole of around 80 D, (69,70) as seen in Figure 1a.…”

Controlling orientation, conformation, and biorecognition of proteins on silane monolayers, conjugate polymers, and thermo-responsive polymer brushes: investigations using TOF-SIMS and principal component analysis

“…TOF-SIMS was also applied to determine the orientation of other functional proteins, mainly immobilized on molecular (i.e., SAM modified) surfaces. The surface orientation of proteins widely applied in bioaffinity techniques such as streptavidin [77] (on polymer layer) and protein G [72,78] (on SAMs) was examined using TOF-SIMS. Relations between orientation and electrostatic interactions as well as biorecognition efficiency were reported.…”

“…Conjugated backbones and alkyl side chains, accessed by TOF-SIMS, correspond to the amorphous morphology of P3ATs and to the edge-on textured crystallites of RP3ATs, respectively. Such a crystalline order (increasing along with the series P3BT < RP3HT < RP3BT < PQT12 [35,77]) can affect protein conformation ( Fig. 5) and improve the electrostatic interactions that control protein orientation (Figs.…”

“…7), adsorbed to two series of solution-deposited polymers with an increasing crystalline order, P3BT < RP3HT < RP3BT [35] (Fig. 6) and P3BT < RP3HT < PQT12 [77] (Fig. 7).…”

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development

“…ToF-SIMS, with sensitivity enhanced for the outermost region of the protein layer, probes surface amino acid concentration, enabling examination of protein orientation in a more direct manner than other surface analysis methods [36,37]. ToF-SIMS analysis of dominant orientation of surfaceimmobilized proteins is well-established for IgG antibody [38][39][40][41][42] and demonstrated also for other proteins [43][44][45][46]. Therefore, we performed PCA of ToF-SIMS data to provide an insight into the orientation of LACT immobilized following two approaches, namely, covalent binding to a GAmodified surface and site-directed binding to an αvβ3 integrin-functionalized surface.…”