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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 edit 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 hydrogen bonds directly made to residues in the binding site the so called first shell of hydrogen bonding involving residues asn23 tyr43 ser27 ser45 asn49 ser88 thr90 and asp128 there is also a second shell of hydrogen bonding involving residues that interact with the first shell residues however the streptavidin biotin affinity exceeds that which would be predicted from the hydrogen bonding interactions alone suggesting another mechanism contributing to the high affinity 4 the biotin binding pocket is hydrophobic and there are numerous van der waals force mediated contacts and hydrophobic interactions made to the biotin when in the pocket which is also thought to account for the high affinity in particular the pocket is lined with conserved tryptophan residues lastly biotin binding is accompanied by the stabilisation of a flexible loop connecting β strands 3 and 4 l3 4 which closes over the bound biotin acting like a lid over the binding pocket and contributing to the extremely slow biotin dissociation rate most attempts at mutating streptavidin result in a lowered biotin binding affinity which is to be expected in such a highly optimized system however an 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 fluorophores flavidin is a streptavidin mutant without lysine side chains which retains good biotin binding characteristics after such fluorescent dye labeling where the dye couples to the amino terminus 6 uses in biotechnology edit 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 nanobiotechnology the use of biological molecules such as proteins or lipids to create nanoscale devices structures in this context streptavidin can be used as a building block to link biotinylated dna molecules to create single walled carbon nanotube scaffolds 8 or even complex dna polyhedra 9 the tetrameric streptavidin has also been used as a hub around which other proteins may be arranged either by an affinity tag such as strep tag or avitag or by genetic fusion to spytag 10 fusion to spytag allowed generation of assemblies with 8 or 20 streptavidin subunits as well as a molecular force probe for atomic force microscopy studies 11 novel materials such as 3d crystalline lattices 12 have also been created streptavidin has a mildly acidic isoelectric point pi of 5 but a recombinant form of streptavidin with a near neutral pi is also commercially available 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 edit monovalent vs monomeric schematic comparing monovalent and monomeric streptavidin streptavidin is a tetramer and each subunit binds biotin with equal affinity multivalency is an advantage in applications like mhc tetramer staining where avidity effects improve the ability of mhc molecules attached to streptavidin to detect specific t cells 14 in other cases such as the use of streptavidin for imaging specific proteins on cells multivalency can perturb the function of the protein of interest monovalent streptavidin is an engineered recombinant form of streptavidin which is a tetramer but only one of the four binding sites is functional this single binding site has 10 14 mol l affinity and cannot cause cross linking 15 applications of monovalent streptavidin have included fluorescent tracking of cell surface receptors decorating dna origami and acting as a pointer to identify specific regions for cryo electron microscopy 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 7 mol l 10 8 mol l and so are not ideal for labeling applications but are useful for purification where reversibility is desirable 16 17 divalent slice through protein comparing biotin orientation in cis divalent and trans divalent streptavidin a streptavidin with exactly two biotin binding sites per tetramer can be produced by refolding together from denaturant of subunits with and without a functional biotin binding site and then purification of the folded tetramers by ion exchange chromatography the functional binding sites here have the same biotin binding stability as wild type streptavidin divalent streptavidin with the two biotin binding sites together cis divalent or apart trans divalent can be separately purified 18 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 edit 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 k d 10 15 m 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 low nonspecific binding of streptavidin see also edit protein tag references edit green nm 1975 avidin advances in protein chemistry 29 85 133 doi 10 1016 s0065 3233 08 60411 8 isbn 9780120342297 pmid 237414 cite journal cs1 maint periodical has isbn link hendrickson wa pähler a smith jl satow y merritt ea phizackerley rp april 1989 crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation proceedings of the national academy of sciences of the united states of america 86 7 2190 4 doi 10 1073 pnas 86 7 2190 pmc 286877 pmid 2928324 weber pc ohlendorf dh wendoloski jj salemme fr january 1989 structural origins of high affinity biotin binding to streptavidin science 243 4887 85 8 doi 10 1126 science 2911722 pmid 2911722 dechancie j houk kn may 2007 the origins of femtomolar protein ligand binding hydrogen bond cooperativity and desolvation energetics in the biotin strept avidin binding site journal of the american chemical society 129 17 5419 29 doi 10 1021 ja066950n pmc 2527462 pmid 17417839 chivers ce crozat e chu c moy vt sherratt dj howarth m may 2010 a streptavidin variant with slower biotin dissociation and increased mechanostability nature methods 7 5 391 3 doi 10 1038 nmeth 1450 pmc 2862113 pmid 20383133 jacobsen mt fairhead m fogelstrand p howarth m august 2017 amine landscaping to maximize protein dye fluorescence and ultrastable protein ligand interaction cell chemical biology 24 8 1040 1047 e4 doi 10 1016 j chembiol 2017 06 015 pmc 5563079 pmid 28757182 holmberg a blomstergren a nord o lukacs m lundeberg j uhlén m february 2005 the biotin streptavidin interaction can be reversibly broken using water at elevated temperatures electrophoresis 26 3 501 10 doi 10 1002 elps 200410070 pmid 15690449 s2cid 16058388 ostojic gn hersam mc june 2012 biomolecule directed assembly of self supported nanoporous conductive and luminescent single walled carbon nanotube scaffolds small 8 12 1840 5 doi 10 1002 smll 201102536 pmid 22461319 zhang c tian c guo f liu z jiang w mao c april 2012 dna directed three dimensional protein organization angewandte chemie 51 14 3382 5 doi 10 1002 anie 201108710 pmid 22374892 fairhead m veggiani g lever m yan j mesner d robinson cv dushek o van der merwe pa howarth m september 2014 spyavidin hubs enable precise and ultrastable orthogonal nanoassembly journal of the american chemical society 136 35 12355 63 doi 10 1021 ja505584f pmc 4183622 pmid 25111182 kim m wang cc benedetti f marszalek pe february 2012 a nanoscale force probe for gauging intermolecular interactions angewandte chemie 51 8 1903 6 doi 10 1002 anie 201107210 pmc 3279624 pmid 22253141 sinclair jc davies km vénien bryan c noble me july 2011 generation of protein lattices by fusing proteins with matching rotational symmetry nature nanotechnology 6 9 558 62 doi 10 1038 nnano 2011 122 pmid 21804552 alon r bayer ea wilchek m august 1992 cell adhesive properties of streptavidin are mediated by the exposure of an rgd like ryd site european journal of cell biology 58 2 271 9 pmid 1425765 xu xn screaton gr october 2002 mhc peptide tetramer based studies of t cell function journal of immunological methods 268 1 21 8 doi 10 1016 s0022 1759 02 00196 5 pmid 12213339 howarth m chinnapen dj gerrow k dorrestein pc grandy mr kelleher nl el husseini a ting ay april 2006 a monovalent streptavidin with a single femtomolar biotin binding site nature methods 3 4 267 73 doi 10 1038 nmeth861 pmc 2576293 pmid 16554831 wu sc wong sl june 2005 engineering soluble monomeric streptavidin with reversible biotin binding capability the journal of biological chemistry 280 24 23225 31 doi 10 1074 jbc m501733200 pmid 15840576 lim kh huang h pralle a park s october 2011 engineered streptavidin monomer and dimer with improved stability and function biochemistry 50 40 8682 91 doi 10 1021 bi2010366 pmid 21892837 fairhead m krndija d lowe ed howarth m january 2014 plug and play pairing via defined divalent streptavidins journal of molecular biology 426 1 199 214 doi 10 1016 j jmb 2013 09 016 pmc 4047826 pmid 24056174 dubacheva galina v araya callis carolina geert volbeda anne fairhead michael codée jeroen howarth mark richter ralf p 9 march 2017 controlling multivalent binding through surface chemistry model study on streptavidin journal of the american chemical society 139 11 4157 4167 doi 10 1021 jacs 7b00540 pmc 5364436 pmid 28234007 fairhead michael veggiani gianluca lever melissa yan jun mesner dejan robinson carol v dushek omer van der merwe p anton howarth mark 21 august 2014 spyavidin hubs enable precise and ultrastable orthogonal nanoassembly journal of the american chemical society 136 35 12355 12363 doi 10 1021 ja505584f pmc 4183622 pmid 25111182 further reading edit hutchens tw porath jo september 1987 protein recognition of immobilized ligands promotion of selective adsorption clinical chemistry 33 9 1502 8 doi 10 1093 clinchem 33 9 1502 pmid 3621554 chodosh la buratowski s 2001 purification of dna binding proteins using biotin streptavidin affinity systems current protocols in protein science vol chapter 9 9 7 1 9 7 13 doi 10 1002 0471140864 ps0907s12 isbn 978 0 471 14086 3 pmid 18429216 s2cid 23660759 zimmermann rm cox ec february 1994 dna stretching on functionalized gold surfaces nucleic acids research 22 3 492 7 doi 10 1093 nar 22 3 492 pmc 523609 pmid 8127690 external links edit 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 howarth lab university of cambridge hytönen lab university of tampere park lab university at buffalo stenkamp lab university of washington wong lab university of calgary retrieved from 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