Distinct Binding Properties of Neutravidin and Streptavidin Proteins to Biotinylated Supported Lipid Bilayers: Implications for Sensor Functionalization

Abstract

The exceptional strength and stability of noncovalent avidin-biotin binding is widely utilized as an effective bioconjugation strategy in various biosensing applications, and neutravidin and streptavidin proteins are two commonly used avidin analogues. It is often regarded that the biotin-binding abilities of neutravidin and streptavidin are similar, and hence their use is interchangeable; however, a deeper examination of how these two proteins attach to sensor surfaces is needed to develop reliable surface functionalization options. Herein, we conducted quartz crystal microbalance-dissipation (QCM-D) biosensing experiments to investigate neutravidin and streptavidin binding to biotinylated supported lipid bilayers (SLBs) in different pH conditions. While streptavidin binding to biotinylated lipid receptors was stable and robust across the tested pH conditions, neutravidin binding strongly depended on the solution pH and was greater with increasingly acidic pH conditions. These findings led us to propose a two-step mechanistic model, whereby streptavidin and neutravidin binding to biotinylated sensing interfaces first involves nonspecific protein adsorption that is mainly influenced by electrostatic interactions, followed by structural rearrangement of adsorbed proteins to specifically bind to biotin functional groups. Practically, our findings demonstrate that streptavidin is preferable to neutravidin for constructing SLB-based sensing platforms and can improve sensing performance for detecting antibody–antigen interactions.

Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding

“…The assay was developed using neutravidin coated SPR chips. Neutravidin is a non-glycosylated analogue of avidin (32) and allows for capture and immobilization of biotinylated ligands onto the SPR chip. Accordingly, rPR3 was biotinylated with NHS-PEG4-biotin which reacts with primary amines such as the side chain of lysine residues or the amino-termini of polypeptides.…”

pH-Modulated Nanoarchitectonics for Enhancement of Multivalency-Induced Vesicle Shape Deformation at Receptor-Presenting Lipid Membrane Interfaces

“…Since the binding strength of the biotin−streptavidin interaction is similarly high across the tested range of pH conditions, the effects of solution pH on multivalency-induced vesicle shape deformation are related to pH-related changes in the membrane properties of vesicles rather than due to changes in the multivalent binding energy per biotin−streptavidin pair. 41,42 Indeed, past results indicate that the biotin− streptavidin binding interaction is mainly mediated by the van der Waals force along with extensive hydrogen bonding rather than by electrostatic forces 42,43 (see also Table S1 for typical pK a values of amino acid side chains involved in the hydrogen bonds). It should also be noted that the effect of solution pH itself on the absolute extent of vesicle deformation is somewhat modest, which is consistent with the tendency for flat PC lipid bilayers to have similar levels of membrane bending rigidity scale-wise in various pH conditions.…”

Real-time detection of HIV-1 using an absorbance-based biosensor: a comparative study of surface coatings and antibody functionalization

“…In this study, an optical biosensor is used to measure the absorbance through a glass slide (with a metal layer coating) which was functionalized with GMBS and Neutravidin. The absorbance is compared when the glass slide is uncoated and coated with Au and Ti using Saline for linkage to protein, Neutravidin protein has binding sites to bind with biotin, and the analysis is done based on time while the HIV-1 DNA is introduced into immobilized surface [8], [9]. The cleaned and activated glass slide was salinized with (3-glyxidyloxypropyl)trimethoxysilane (GPTM) and the activated Au and Ti slide were functionalized with (N-γ-maleimidobutyryloxysuccinimide ester) GMBS [10].…”