Elucidating the Binding Mechanism of a Novel Silica-Binding Peptide

Abstract

Linker-protein G (LPG) is a bifunctional fusion protein composed of a solid-binding peptide (SBP, referred as the "linker") with high affinity to silica-based compounds and a Streptococcus protein G (PG), which binds antibodies. The binding mechanisms of LPG to silica-based materials was studied using different biophysical techniques and compared to that of PG without the linker. LPG displayed high binding affinity to a silica surface (K D = 34.77 ± 11.8 nM), with a vertical orientation, in comparison to parent PG, which exhibited no measurable binding affinity. Incorporation of the linker in the fusion protein, LPG, had no effect on the antibody-binding function of PG, which retained its secondary structure and displayed no alteration of its chemical stability. The LPG system provided a milder, easier, and faster affinity-driven immobilization of antibodies to inorganic surfaces when compared to traditional chemical coupling techniques.

The Effect of Oligomerization on A Solid-Binding Peptide Binding to Silica-Based Materials

“…Analysis of the orientation of 3 × LPG on the silica surface was determined by taking into account its molecular weight of 28.2 kDa and globular nature, as performed previously for 4 × LPG [ 34 ], and was approximated to be 324 and 175 ng/cm 2 for end-on and side-on orientation, respectively. The mass of 3 × LPG on the silica surface was approximately 680 ng/cm 2 ( Table 3 ), which, based on the analysis above, is most likely a hydrated end-on configuration, as observed previously for 4 × LPG [ 31 ]. Based on these results, it appears that the three repeats of the linker (3 × LPG) support a similar level of binding as 4 × LPG, but with comparatively weaker binding affinity.…”

“…The mass of trastuzumab immobilized to 3 × LPG was approximately 1000 ng/cm 2 to which approximately 1250 ng/cm 2 HER2 antigen was immobilized ( Table 4 ). These results indicated less immobilization than reported for 4 × LPG [ 31 ]. These results are in accordance with the stoichiometric data presented above, where as many as twice the number of 4 × LPG molecules were bound on the silica surface per cm 2 as compared to 3 × LPG.…”

“…The sensorgram for 2 × LPG and a silica surface ( Figure 4 b) showed measurable binding at the three highest protein concentrations (654, 65.4, and 13.1 nM). Although it was not possible to calculate the binding affinity for 2 × LPG, the frequency shift for the highest 2 × LPG concentration suggests that the adsorption of the protein to the silica surface is almost half (~16.5 Hz) that previously reported observed for 4 × LPG [ 31 ]. Binding of 3 × LPG to silica was observed at all concentrations analysed with both changes in f and D measurements observed ( Figure 4 c,d) and a binding affinity (K D ) of 53 ± 5 nM, when fitted using the Langmuir isotherm ( Figure S3 , Supplementary Materials ).…”

“…The concentration of GdnHCl required for 50% unfolding of the core protein where the tryptophans are located was determined to be 3.4 ± 0.2 M (95% CI), 3.4 ± 0.1 (95% CI), and 3.4 ± 0.2 (95% CI) for 1 × LPG, 2 × LPG, and 3 × LPG, respectively. The concentrations for PG and 4 × LPG were previously reported as 3.4 ± 0.2 M and 3.6 ± 0.1 M, respectively [ 31 ]. Thus, the chemical stability of the core PG portion of the fusion proteins is, within experimental error, unaffected by the linker sequences.…”

“…All solutions were passed through a 0.22 μm filter and degassed at room temperature before use. The adsorbed mass is proportional to the change in f (∆ f ) via the Sauerbrey equation only when the adsorbed layer is rigid, uniformly distributed on the sensor surface, and the adsorbed mass is small compared to the mass of the crystal [ 31 , 32 ]. However, in the case of most proteins, these assumptions are not valid, as the adsorbed layer is viscoelastic as there is a dissipation change greater than 1 × 10 6 /10 Hz.…”