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Revision as of 14:23, 10 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[cite]. 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.
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[cite].
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°. Trp120, which binds the biotin molecule from an adjacent subunit, displays a much larger influence in binding free energy[cite].
Moreover the hydrogen bonding network and van der waals interactions show great influence in binding free energy. Compared to similar hydrogen bonding donors and acceptors of other protein-ligand systems the hydrogen bonding of Streptavidin to the ureido oxygen of biotin is remarkably high.
The activation barrier for native streptavidin is due to a large activation enthalpy of +32 kcal/mol at room temperature. The activation entropy is 7.6 kcal/mol (22 °C). Weiter 4 : 41
Streptavidin variantes and homologes
Traptavidin
The streptavidin mutant (S52G, R53D) shows a 10 times lower dissociation constant (kD= 10-16 M) and increased mechanical strength of the biotin binding. Moreover, it had improved thermostability before splitting in monomers (~10 °C higher)[cite]. In contrast to streptavidin, the Traptavidin L3/4 loop (residues 45-50) does not change its conformation while binding biotin. figure oben The binding pocket is already closed and lacks flexibility. The loss of a structural change may decrease the entropic cost of binding and inhibits dissociation. The traptavidin dissociation is faster at pH 5 than at pH 7.4, but still it is significantly slower than streptavidin dissociation. The hydrogen bonding length of both proteins to biotin are comparable.
Strep-Tactin
Strep-Tactin[cite] is an engineered streptavidin variant, which can bind a specific peptide sequence, called Strep tag. The Peptide sequence is eight residues long and can be fused N- or C-terminal to recombinant protein by subcloning. Using the Strep tag-Strep-Tactin-System in affinity chromatography provides great yields in protein- or protein complex purification. The strep-tagged protein binds Strep-Tactin, which is immobilized on the column. After washing, the protein can be eluted with biotin, which binds the Strep-Tactin several orders of magnitude stronger. The Strep tag (amino acid sequence: WRHPQFGG) was primarily designed to bind Streptavidin, over the years both the Strep tag and the Streptavidin were optimized. Current state of the art is strep tag II (WSHPQFEK) binding to Strep-Tactin. Strep Tag II is able to bind recombinant protein at more attachment sites than its precursor. It is rarely degraded by cellular proteases and biochemically almost inert. Thus, it does not affect the protein folding or secretion.
enhanced monomeric Avidin (eMA)
eMA was engineered for applications like molecular labelling without unwanted cross-linking of biotin conjugats. The monomeric protein, however, does not consist of a monomeric streptavidin subunit. Streptavidin binding pockets depend on one residue from a adjacent subunit, thus binding affinity of a single subunit is decreased (KD = 10-7-10-9 M). eMA consists of a monomerized Rhizavidin dimer, which contains a disulfid bond in the binding site restraining the protein and forming a rigid binding site[cite].
single chain avidin
...
- ↑ Schmidt, T. G., & Skerra, A. (2007). The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nature protocols, 2(6), 1528-1535.
