Streptavidin
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UniProt P22629 | | ||||||
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Streptavidin
Structure
The crystal structure of streptavidin with biotin bound was reported by two groups in 1989. The structure was solved using multi wavelength anomalous diffraction by Hendrickson et al.[2] at Columbia University and using multiple isomorphous replacement by Weber et al.[3] at E. I. DuPont Central Research and Development Department. As of September 2017, there are 171 structures deposited in the Protein Data Bank. See this link for a complete list. The N and C termini of the 159 residue full-length protein are processed to give a shorter ‘core’ streptavidin, usually composed of residues 13–139; removal of the N and C termini is necessary for the highest biotin-binding affinity. The secondary structure of a streptavidin monomer is composed of eight antiparallel β-strands, which fold to give an antiparallel β-barrel tertiary structure. A biotin binding-site is located at one end of each β-barrel. Four identical streptavidin monomers (i.e. four identical β-barrels) associate to give streptavidin's tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from a neighboring subunit. In this way, each subunit contributes to the binding site on the neighboring subunit, and so the tetramer can also be considered a dimer of functional dimers.
Origins of the high affinity
The numerous crystal structures of the streptavidin-biotin complex have shed light on the origins of the remarkable affinity. Firstly, there is high shape-complementarity between the binding pocket and biotin. Secondly, there is an extensive network of hydrogen bonds formed to biotin when in the binding site. There are eight
Most attempts at mutating streptavidin result in a lowered biotin-binding affinity, which is to be expected in such a highly optimized system. However, a recently engineered mutant of streptavidin, named traptavidin, was found to have more than ten-fold slower biotin dissociation, in addition to higher thermal and mechanical stability.[5] This decreased dissociation rate was accompanied by a two-fold decrease in the association rate.
Biotin-binding affinity can be impaired by chemical labeling of streptavidin, such as with amine-reactive
Uses in biotechnology
Among the most common uses of streptavidin are the purification or detection of various biomolecules. The strong streptavidin-biotin interaction can be used to attach various biomolecules to one another or onto a solid support. Harsh conditions are needed to break the streptavidin-biotin interaction, which often denatures the protein of interest being purified. However, it has been shown that a short incubation in water above 70 °C will reversibly break the interaction (at least for biotinylated DNA) without denaturing streptavidin, allowing re-use of the streptavidin solid support.[7] A further application of streptavidin is for purification and detection of proteins genetically modified with the Strep-tag peptide. Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase. Streptavidin has also been used in the developing field of
- Pretargeted Immunotherapy
Pretargeted immunotherapy uses streptavidin conjugated to a monoclonal antibody against cancer cell-specific antigens followed by an injection of radiolabelled biotin to deliver the radiation only to the cancerous cell. Initial hurdles involve saturation of the biotin binding sites on streptavidin with endogenous biotin instead of the injected radiolabelled biotin, and a high degree of radioactive exposure in the kidneys due to streptavidin's strong cell adsorptive properties. It is currently thought that this high level of binding to adherent cell types, such as activated platelets and melanomas, is a result of integrin binding mediated through the RYD sequence in streptavidin.[13]
Variants with a controlled number of binding sites
- Monovalent vs. monomeric
Streptavidin is a tetramer and each subunit binds biotin with equal affinity. Multivalency is an advantage in applications like
Monomeric streptavidin is a recombinant form of streptavidin with mutations to break the tetramer into a monomer and to enhance the solubility of the resultant isolated subunit. Monomeric streptavidin versions have an affinity for biotin of 10−7mol/L 10−8mol/L and so are not ideal for labeling applications but are useful for purification, where reversibility is desirable.[16][17]
- Divalent
A streptavidin with exactly two biotin binding sites per tetramer can be produced by mixing subunits with and without a functional biotin binding site and purification by
- Trivalent
A streptavidin with exactly three biotin binding sites per tetramer can also be produced using the same principle as to produce divalent streptavidins.[19]
- High-valency streptavidins
Streptavidins of higher valency has been obtained by utilizing the chemistry of isopeptide bond conjugation using the SpyTag/SpyCatcher technology.[20] This involves having a streptavidin tetramer with three biotin binding sites and a dead streptavidin fused to either SpyTag or SpyCatcher. When the different tetramers are mixed together, a covalent linkage occurs to enable higher number of biotin binding sites. Six and twelve biotin binding sites per molecule have been made with this method.
Comparison to avidin
Streptavidin is not the only protein capable of binding to biotin with high affinity. Avidin is the other most notable biotin-binding protein. Originally isolated from egg yolk, avidin only has 30% sequence identity to streptavidin, but almost identical secondary, tertiary and quaternary structure. Avidin has a higher affinity for biotin (Kd ~ 10−15M) but in contrast to streptavidin, avidin is glycosylated, positively charged, has pseudo-catalytic activity (avidin can enhance the alkaline hydrolysis of an ester linkage between biotin and a nitrophenyl group) and has a higher tendency for aggregation. On the other hand, streptavidin is the better biotin-conjugate binder; avidin has a lower binding affinity than streptavidin when biotin is conjugated to another molecule, despite avidin having the higher affinity for free, unconjugated biotin. Because streptavidin lacks any carbohydrate modification and has a near-neutral pI, it has the advantage of much lower nonspecific binding than avidin. Deglycosylated avidin (NeutrAvidin) is more comparable to the size, pI, and nonspecific binding of streptavidin.
See also
References
Further reading
- Hutchens TW, Porath JO (September 1987). "Protein recognition of immobilized ligands: promotion of selective adsorption". Clinical Chemistry. 33 (9): 1502–8. PMID 3621554.
- Chodosh LA, Buratowski S (2001). "Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems". S2CID 23660759.
- Zimmermann RM, Cox EC (February 1994). "DNA stretching on functionalized gold surfaces". Nucleic Acids Research. 22 (3): 492–7. PMID 8127690.
External links
- Swiss-Prot entry for Streptavidin precursor from Streptomyces avidinii
- Streptavidin at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
- Egg-stremely useful interaction QUite Interesting PDB Structure article at PDBe
Groups investigating and developing streptavidin or avidin-family proteins (Alphabetical order)