Wild Type Core Streptavidin
Nicholas Gutsche '17 and Jack Clayton '17
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
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,
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.
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-(4í-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
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 itís 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 itís 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
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
Streptavidin. The Journal of
Biological Chemistry 26:12728-12729.
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