Bifunctional Avidin with Covalently Modifiable Ligand Binding Site

Abstract

The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

Multifunctional streptavidin–biotin conjugates with precise stoichiometries

“…4424 | Chem. Sci., 2020, 11,[4422][4423][4424][4425][4426][4427][4428][4429] This journal is © The Royal Society of Chemistry 2020 where atto-565 absorbs (Fig. S4a, † relative integrated areas at 280 nm: 5.1% 1 st peak, 42.6% 2 nd peak, 26.7% 3 rd peak and 25.6% 4 th peak).…”

“…[6][7][8][9][10] Alternatively, (strep)avidins composed of different subunits have been used to integrate different functionalities within one conjugate. 11,12 These streptavidins have been used to form structurally dened one-toone streptavidin-biotin conjugates and image biotinylated cell surface receptors without the formation of articial receptor clustering. However, the monovalent streptavidins are unt as linkers to assemble streptavidin conjugates with multiple functional groups, or with multiple copies of one functional group.…”

Molecular features of steroid-binding antidins and their use for assaying serum progesterone

“…g . Avds with higher thermal stability [4,6]; single-chain and dual-chain Avds–circularly permuted Avds consisting of four or two monomers, respectively, fused into a single polypeptide chain–that have potential to bind simultaneously up to four different ligands as well as monomeric avidins, enabling applications where oligomeric assembly is disadvantageous [5,7–11]. Despite intensive research focusing on Avd structure and function, the biological role of Avd remains partially unclear.…”

Mixture of PLA–PEG and biotinylated albumin enables immobilization of avidins on electrospun fibers

“…Binding between biotin and avidin is very tight, and several studies have demonstrated that usually sufficient conditions for protein denaturation are failing to weaken the binding between avidin and biotin complexes . It is also possible to independently control individual binding sites and alter their affinity for the ligands . Once the avidin–biotin complex is formed, it is unaffected by wide extremes of pH (between 2 and 13), high temperature (melting point around 120°C), organic solvents, or other denaturing agents.…”

“…28 It is also possible to independently control individual binding sites and alter their affinity for the ligands. 29 Once the avidin-biotin complex is formed, it is unaffected by wide extremes of pH (between 2 and 13), high temperature (melting point around 1208C), organic solvents, or other denaturing agents. Avidin retains its ability to bind to biotin at room temperature (RT) in the presence of common surfactant such as SDS, SDBS, and Triton X-100.…”