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[[File:Muc16_Proteins_Apo-streptavidin_and_streptavidin-biotin_complex.jpg |frame|center|450px| <i><b>Figure 2:</b></i> Apo-streptavidin and streptavidin-biotin complex<ref>Chivers, C. E., Crozat, E., Chu, C., Moy, V. T., Sherratt, D. J., & Howarth, M. (2010). A streptavidin variant with slower biotin dissociation and increased mechanostability. Nature methods, 7(5), 391-393.</ref>]]
 
[[File:Muc16_Proteins_Apo-streptavidin_and_streptavidin-biotin_complex.jpg |frame|center|450px| <i><b>Figure 2:</b></i> Apo-streptavidin and streptavidin-biotin complex<ref>Chivers, C. E., Crozat, E., Chu, C., Moy, V. T., Sherratt, D. J., & Howarth, M. (2010). A streptavidin variant with slower biotin dissociation and increased mechanostability. Nature methods, 7(5), 391-393.</ref>]]
  
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Important for the high barrier of dissociation are biotin-tryptophan contacts in the binding pocket. There are four significant tryptophan residues involved in the binding site. The three residues Trp79, Trp92 and Trp108 are lined up in one section. Site directed mutagenesis states that W79F and W108F show small ΔΔG°.
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Important for the high barrier of dissociation are biotin-tryptophan contacts in the binding pocket. There are four significant tryptophan residues involved in the binding site. The three residues Trp79, Trp92 and Trp1
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Trp120, which binds the biotin molecule from an adjacent subunit, displays a much larger influence in binding free energy<ref>Stayton, P. S., Freitag, S., Klumb, L. A., Chilkoti, A., Chu, V., Penzotti, J. E., ... & Stenkamp, R. E. (1999). Streptavidin–biotin binding energetics. Biomolecular engineering, 16(1), 39-44.</ref>.
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Revision as of 22:48, 11 October 2016


Streptavidin – Biotin

General

Streptavidin is a tetrameric protein with a molecular weight of 4 x 13,33 kDa[1], it can be isolated from Streptomyces avidinii. Each subunit is able to bind one molecule of biotin (molecular weight = 244.3 Da). This specific, non-covalent bondage with a femto molar dissociation constant (kD = 10-15 M) is one of the strongest known biological affinities. Antibodies, in comparison, have lower dissociation constants with 10-7 – 10-11 M.

One subunit of streptavidin is organized as eight stranded antiparallel beta sheets of coiled polypeptide chains which form a hydrogen bonded barrel with extended hydrogen loops[2]. The biotin binding pocket consists of primarily aromatic or polar side chains. These interact with the hetero atoms of biotin.

Due to its low unspecific binding, Streptavidin is primarily used in protein-purification and imaging. Biotinylation does not interrupt functions of a biomolecule. Biotin ligases can attach biotin to specific lysine residues in vitro and in vivo.

Figure 1: Streptavidin Tetramer and Monomer

Application in our project

We use the extraordinary strength of the biotin:streptavidin binding for linking genetically engineered cells with streptavidin. The cells express a receptor which allows them to present a biotin molecule on their cell surface (see receptor construct).

The tetrameric Protein itself is able to cross link the biotinylated cells and form a stable network. A linker molecule like PAS-Lysin, however, might increase the stability of the cell structure (see PAS).

Reasons for high affinity

The rapid and irreversible linkage is generally due to multiple hydrogen bonds as well as van der Waals interactions. Comparing the structures of apostreptavidin and the streptavidin-biotin complex, determined by multiple isomorphous replacement, provides several structural differences of the binding pocket.

The ordering of two flexible surface polypeptide loops results in burying the biotin molecule in its pocket. The L3/4 loop near biotins valeryl tail is typically disordered, but closes upon biotin binding[3].

Figure 2: Apo-streptavidin and streptavidin-biotin complex[4]
Important for the high barrier of dissociation are biotin-tryptophan contacts in the binding pocket. There are four significant tryptophan residues involved in the binding site. The three residues Trp79, Trp92 and Trp1
  1. ↑ http://www.expasy.org/
  2. ↑ Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., & Salemme, F. R. (1989). Structural origins of high-affinity biotin binding to streptavidin. Science, 243(4887), 85.
  3. ↑ Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., & Salemme, F. R. (1989). Structural origins of high-affinity biotin binding to streptavidin. Science, 243(4887), 85.
  4. ↑ Chivers, C. E., Crozat, E., Chu, C., Moy, V. T., Sherratt, D. J., & Howarth, M. (2010). A streptavidin variant with slower biotin dissociation and increased mechanostability. Nature methods, 7(5), 391-393.