Wild Type Core Streptavidin

Nicholas Gutsche '17 and Jack Clayton '17

Contents:

I. Introduction

A protein isolated from the bacterium Streptomyces avidinii, streptavidin is commonly identified and known for its particularly strong binding affinity to biotin (Vitamin H). This binding interaction is said to be one of the strongest non-covalent interactions in nature, with a binding affinity constant of 10^13 M^-1. Due to this affinity, biotin-streptavidin complexes have diverse applications in protein and nucleic acid detection and purification methods (Western, Southern, Northern blotting, Immunoprecipitation, FACS and EMSA to name a few). This high binding affinity also ensures that once formed, the bond between biotin and streptavidin is extremely resistant to extremes of pH, temperature, organic solvents, and other denaturing agents.

Most streptavidin-biotin detection methods involve the application of biotinylated probes which interact with the target molecule before binding to labeled streptavidin. The combination of the stability of the biotin label, and the molecule's relatively small size makes it an attractive marker in detection assays.

II. General Structure

Streptavidin is a tetramer of about 52,000 daltons (four 13,000 dalton subunits). Identical subunits A and B are on a diagonal plane to each other, and each subunit is an eight stranded beta barrel with a biotin binding site at the end of the barrel. In addition, various subunits interact with each other, contributing molecular interaction via a conserved tryptophan residue. In this way, the tetramer can be thought of as a dimer of dimers. To contribute the particular tryptophan residue to the opposite subunit's binding site, an extended loop reaches from one subunit to another (across the diagonal mirror plane).

III. Biotin Binding



Each streptavidin tetramer binds four molecules of biotin. The binding of biotin to the protein involves three major components. The first is hydrophobic and van der Waals interactions between the biotin and various tryptophan molecules: W-79, W-92, W-108, and W-120. The second are side-chain hydrogen bonding interactions between the streptavidin residues and various biotin atoms occurring at Asp-128, Asn-23, Ser-27, Ser-45, and Tyr-43. The last component is the interaction of the surface loop connecting beta strands 3 and 4. This loop can adopt "open" and "closed" confirmations. When biotin is bound to streptavidin, the loop adopts the "closed" confirmation, further strengthening the binding interaction.

IV. Biotinylation

Biotinylation is the process of covalently attaching a biotin molecule to a protein, nucleic acid or other molecule. As we have seen, biotin has a very high binding affinity for streptavidin. This process takes advantage of that fact by binding a molecule of biotin to a molecule of interest and adding streptavidin, which will bind to that molecule with a high affinity in order to isolate it. This process is useful in determining protein-protein interactions, purification methods, etc. In order to assess the extent of biotinylation, researchers use the organic dye, HABA [2-(4’-hydroxyphenylazo) benzoic acid]. HABA utilizes the same binding site in streptavidin as biotin and has an identifiable absorbance. However, when biotinylated molecules are introduced, HABA is displaced and a proportional change in absorbance can be recorded.

HABA has a much lower binding affinity (Ka = 104 M^-1) to streptavidin compared to biotin (Ka = 1013 M^-1). HABA interacts with the binding site similarly to biotin, with it’s benzoate oxygen lining up where the ureido oxygen of biotin would be found. It also forms H-bonds with Asn-23, Tyr-43 and Ser-27. HABA binds as a hydrazone tautomer so that the adjacent nitrogen can donate a hydrogen to stabilize the other oxygen of the benzoate. This second oxygen also H-bonds with Ser-45, switching it to a donor instead of an acceptor (as it is with biotin). HABA is unable to bind to Asp-128, Ser-88 or Asn-49 which affects it’s enthalpic stability (Delta H = 1.70 kcal/mol). The greatest difference from the binding affinity of biotin is the loss of charge localization that biotin (Delta H = -32.0 kcal/mol) has on the ureido oxygen (negative) and nitrogens (positive).

V. References

Le Trong, Isolde, Zhizhi Wang,David E. Hyre, Terry P. Lybrand, Patrick S. Stayton and Ronald E. Stenkamp. 2011. DNA Streptavidin and its biotin complex at atomic resolution. Acta Cryst. D67:813-821

Weber, P. C., J. J. Wendeloski, M.W. Pantoliano, and F.R. Salemme. 1992. Crystallographic and Thermodynamic Comparison of Natural nd Synthetic Ligands Bound to Streptavidin.  J.Am.Chem.Soc 114: 3197-3200.

Weber, P. C., M. Jane Cox,  F. Raymond Salemme, and Douglas H. Ohlendorf. 1987. Crystallographic Data for Streptomyces avidinii Streptavidin. The Journal of Biological Chemistry 26:12728-12729.

Back to Top