© 2010 Nature America, Inc. All rights reserved. 1 stability streptavidin-biotin in decrease surprising a cause days ~4 of a lifetime had est inter of receptor the 2 h, in whereas detected being of streptavidin dissociation to led pH endosomal low imaging, in example, is biotin is irreversible interaction essentially far from correct. For available. commercially are conjugates biotin and streptavidin animals using biotin ligase proteins to targeted precisely be can biotin HaloTagligands, and SNAP-tag unlike Also, binding. proteins may and binding promote unfold, nonspecific aggregate covalent- these conditions, harsh these under attached remain ture, pH or and will so ligand denaturant, prebound even though tempera to streptavidin of resistance the have not do domains their and ligand are for valuable cellular labeling to bonds covalent irreversible HaloTag form SNAP-tag, or using those as such methods, targeting Alternative bio function. molecule disrupt not does generally biotinylation and binding cific have and biotin low nonspe Streptavidin trials. in clinical cancer success showing is and nano-assembly, and purification protein most stably to biotin conjugates the binds streptavidin but species, of range wide a from isolated widely is exploited in biological(neutr)avidin research or streptavidin proteins the and biotin molecule small the between interaction strong remarkably The force generated by DNA segregation, rapidly displaced streptavidin from biotinylated applications. this thermostability; resilience should enable diverse increased dissociation, mechanical strength and improved streptavidin, traptavidin, with more than tenfold slower biotin andamplification nanotechnology. Weidentified mutant a but dissociation does occur, limiting its use in imaging, S Vincent T Moy Claire E Chivers mechanostability dissociation and increased with slower biotin A Received Received 7 Oct Florida, USA. Correspondence should be addressed to M.H. ([email protected]). Department of Biochemistry,of Department Oxford University, Oxford, UK. treptavidin treptavidin binds biotin conjugates with exceptional stability Despite its stable binding, the perception that the the its binding, streptavidin- Despite stable perception streptavidin variant , , whereas traptavidin resisted indicating displacement, the o be F tsK, tsK, a motor protein important in chromosome r 2009; accepted 5 Ma 2 , David J Sherratt 1 F , Estelle Crozat tsk tsk translocation. 1 . An additional advantage is that many 3 . Also, nanoparticle attachment can can attachment . Also, nanoparticle 1 . Biotin-binding proteins have been 1 . Streptavidin . is Streptavidin used in imaging, r in vitro in ch ch 2010; published 1 , ChuCalvin 1 & Mark Howarth , on cells and in living living in and cells on , 2 . However, these 2 Department of Physiology and of Biophysics,Department University Miami,of Miller Medicine,School of Miami, o nline nline 11 2 ,

DNA A 4 p ; the the ; 1 r

il il 2010; - - - -

cluding cluding use in cells. We therefore aimed to a engineer streptavidin conjugates and in systems unaffected by changing redox state, pre revers controlled ibility giving conjugate, biotin thiol-linked a with dissociation reduce to bis-biotinylated be to DNA has but DNA sequencing, 454 and BEAMing PCR, for as such peratures, DNA along polymerase or DNA RNA polymerase helicases, as such motors molecular of of tens to secondsms 20 of arrests with across, roll instead but surface capillary, streptavidin-coated beads do not attach to a biotinylated beads to attached was streptavidin when million-fold a approximately ( constant dissociation thanstreptavidin dissociation ( dissociation was faster than at pH 7.4 but was stillavidin significantlydissociated slowereven faster than streptavidin subsequenth.12contrast,In streptavidin dissociated steadily, and theover traptavidinfromdissociation little was point,there time jugates ( Traptavidinalso had dramaticallya reduced rate off to biotin con how derivatization at the carboxyl group changes binding strength. action with the carboxyl group of biotin substantial part of (strept)avidin’s binding energy comes from forinter traptavidin and 6.8 tenfold lower for traptavidin than streptavidin (4.2 decrease the association (‘on’) rate and enhance thermostability. the entropic cost of biotin binding and inhibit dissociation as well overas the biotin-binding pocket Fig. 1 (residues45–50;loop L3/4 the flexibilityof reducetraptavidin in mutationstraptavidin.termedtheWe hypothesize thatwe which the lowest off rate for the S52G,R53D mutantaccording of to streptavidinbiotin–4-fluorescein ( dissociation (‘off’) rate and found We randomized promising residues and evaluated purified proteins mutations adjacent to the biotin carboxyl and in the L3/4 loop rings of biotin, which invariably impair binding thisliterature, avoidedwe mutations nearureidothe thiophene or ing as wild-type streptavidin-biotin has been identified with improved desthiobiotin binding, but nocompartmentalization, pair with yielding, foras example, strong a streptavidin variantbind screenedbeenvariousfor properties phage displayby and haveimproved biotin-binding stability ance.Over 200 streptavidin mutants have been described but none conjugate. biotin any to stably more bind would that mutant d o A streptavidin mutant containing a cysteine forms a disulfide disulfide a forms cysteine a containing mutant streptavidin A The off rate for free biotin at 37 °C and pH 7.4 was more thanmore was 7.4 pH and °C 37 at biotinfree for rate off The In a highly optimized system, almost any change reduces perform i : 1 0 a . 5 9 1 ). Upon biotin binding, this loop becomes ordered and closes . In the presence of shear forces lower than those in a blood , but this mutant only enhances binding to certain biotin biotin certain to binding enhances only mutant this ,but 0 6 3 P . In addition, streptavidin cannot prevent the translocation 8 = 0.0008; / n m e t h nature methods . 1 4 5 Fig. 0 ±

0.3 × 10 1 K 7 c . Streptavidin can be used at high tem high at used be can Streptavidin . brief communications d ). After the ~2% dissociation at the initial ) for a biotinylated peptide increased increased peptide biotinylated a for ) 1 P 2 −5

. Amore ordered loop may reduce = 0.001) ( | ADVANCE s −1 for streptavidin; 1 1 .Streptavidin libraries have , it is important to establish

Fig. ONLINE 1 1 . At pH 5, traptavidin , and explored many

1 d ).The on rate of

PUBLICATION ± 0.5 × 10 10,11 Fig. 1 . Based on Fig. 1 in vitro in b 8 1,10,11 ). As a . −6 s | a

−1 ),  ------.

© 2010 Nature America, Inc. All rights reserved. lane is a negative control with no streptavidin or traptavidin added. Bands corresponding to DNA that is free or bound to streptavidin or traptavidin are traptavidin or streptavidin to bound or free is that DNA to corresponding Bands added. traptavidin or no streptavidin with control a negative is lane left The of DNA. imaging fluorescence and electrophoresis gel agarose before temperatures indicated the at 3 min for heated and DNA biotinylated with ( (right). plotted was 2) 1 and as (indicated gels duplicate from of monomers percentage The indicated. are bands (M) monomer and Tetramer (T) °C. 95 at heating before SDS with mixed was (C) control positive The stained. Coomassie and SDS-PAGE by separated then and temperatures, indicated the at 3 min for incubated were Proteins (middle). traptavidin or F altered The tag. 15-amino-acid a with modified be to only needs protein target the and sensitive, and rapid is streptavidin traptavidin. to bound ( still was ligand most temperatures high but streptavidin, from dissociation at complete was °C, there At 70 stability binding conjugate ( streptavidin for than higher °C ~10 was transition of midpoint the monomers; into splitting before streptavidin to compared thermostability increased had means that longer incubations are required to reach equilibrium. reduced ( ( 0.1 × 10 traptavidin for biotin–4-fluorescein was reduced twofold, from 2.0  lane. each under labeled is traptavidin or streptavidin from free DNA of biotinylated percentage The marked. ( streptavidin dissociation rates at pH 5.0, analyzed as in the increase in fluorescence was measured at 37 °C. ( upon addition of excess free biotin, dissociates, biotin–4-fluorescein and The proteins bind to quenching biotin–4-fluorescein, its fluorescence; pH 7.4. ( the fraction of [ to [ ( the L3/4 loop is disordered and does not give clear electron density. of van der Waals radius. Ser52 and Arg53 are colored green. Without biotin, with biotin and(1mk5) without biotin Biotin (1swa). is shown as spheres mutated to produce in traptavidin structures (S52G,R53D) of streptavidin compared to streptavidin. ( F P n b igure igure igure igure

b a brief communications = 3). Some error bars are too small to be visible. ) ) Streptavidin and traptavidin off rates, measured by binding the proteins

= 0.004), whereas the on rate of traptavidin for [ 3 | Imaging of cell-surface proteins using biotin ligase and and ligase biotin using proteins cell-surface of Imaging Traptavidin temperature. high at used often is Streptavidin

ADVANCE In(bound [ H] biotin) 3 Arg53 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 H]biotin, H]biotin, then adding excess nonradioactive biotin and determining 0 a 6 4 2 0 2 1 Strepta 7 with biotin c | | M (kDa ) Dissociation rates of traptavidin, streptavidin and avidin at pH 7.4. Ser5 MW Traptavidin has a dissociation slower rate from biotin conjugates Traptavidin has increased thermostability compared to streptavidin. ( streptavidin. to compared thermostability increased has Traptavidin 10 15 25 35 40 55 70 Supplementary −1 ) vidi 2

s ONLINE −1 L3/ loop

25 n 3 H]biotin H]biotin remaining bound after varying times at 37 °C and for streptavidin to 1.0 Time (h

50 4 Temperature (°C 60

65 Streptavidin

PUBLICATION 70 ) without bioti Strepta

75 Tr 1 8 Strepta

80 apta

85 a ) ) PyMOL images showing the residues

vidin 1 0 Fig. )

90 vidi vidin 95 n

100 n 2

C 1 Fig. ). The slower on rate traptavidinof | T M d c

nature methods Dissociation (%) Dissociation (%) ±

10 15 20 25 10 15 20 2 0.03 × 10 0 5 0 5 a (kDa 0 0 MW 10 15 25 35 40 55 70 ). We also assessed biotin- assessed Wealso ). ) 8 6 4 2 8 6 4 2 25 Tr Strepta apta

50 7 Temperature (°C 60 M vidin d vidin

65 Traptavidin ) ) Traptavidin and −1 Time (h 3 Time (h H]biotin was also 70 c s . . Error bars, 1 s.d.

75 −1 Tr Strepta Av 10 b 80 apta idin for traptavidin ) ) Thermostability of biotin-conjugate binding. Streptavidin or traptavidin were incubated incubated were traptavidin or Streptavidin binding. of biotin-conjugate ) Thermostability 85 ) 12 vidi )

90 vidin

95 Fig. n 14

100 10 16 C

2 T M 18 b 12 ). ±

Monomer (%) 100 10 20 30 40 50 60 70 80 90 0 ical binding stability than streptavidin over a range of loading loading of rates ( range a over streptavidin than stability binding ical atomic force microscopy (AFM). Traptavidin had greater mechan the level at by probed the strength of traptavidin Wesingle-molecule mechanical dissociation. for energy activation the of scape land and height the to force force changes complex: is resistance traptavidin. of rate on slower the with consistent shown), not (data biotin. streptavidin with intense more was staining cell with the tions, comparable was traptavidin streptavidin and ( traptavidin pre-blocked with we Staining when binding minimal and BirA-ER, and AP-IGF1R expressing cells on signal fluorescently strong a with imaging, for with specificity ( high showed Traptavidin streptavidin AP-IGF1R or traptavidin biotinylated labeled detected and (BirA-ER) ligase biotin co-expressed with peptide acceptor the receptor IGF1R to the acceptor (AP-IGF1R), peptide biotinylated in factor streptavidin growth to 1 insulin-like We cells. type the fused mammalian specificity similar had traptavidin whether mutants to lower-pI compared avidin for seen as binding, cellular nonspecific affect may traptavidin of charge within 3 min,whereastraptavidinresisteddisplacement3 ( within 0.5 tering streptavidin or traptavidin bound to the biotin ( the DNA in a defined orientation and then translocate until biotinylatedencoun nucleotide near the end, so that FtsK would load on to directionallyFtsK loads thatsequence KOPS,8-bp an with obstacles in its path, we used a short DNA substrate containing tectural proteins. To study how DNA-bendingfactors,transcriptionand RNApolymerases archi cell. daughter each tochromosome one of partition faithful ensuring and dimers chromosome separating XerC/D, by recombination until encountering XerC and XerD; then FtsK activates site-specific DNAalongtranslocates (refs.divide,FtsK13,14). bacteria Before s kb 5 at DNA along translocating proteins, motor molecular at a force given ( traptavidin and streptavidin between rate dissociation the in difference the mate relationship between loading rate and rupture force, we could esti thermal to fluctuations traversing the activation barrier. From the ( strengths Supplementary a 25 ) Thermostability analysis of tetramer structure of streptavidin (left) (left) of streptavidin structure of tetramer analysis ) Thermostability The relationship between binding stability over time versus versus time over stability binding between relationship The ept te tegh f h srpaii-itn interaction, streptavidin-biotin the of strength the Despite fastest the of one FtsK, study to traptavidin applied We Traptavidin (2 Traptavidin (1 Streptavidin (2 Streptavidin (1 µM 50 Invivo 60 P < 0.0001) ( Temperature FtsKdisplaced streptavidinmajoritythe of fromDNAthe 65 70 Supplementary DNA isbound to many proteins, including repressors, ) ) ) ) 75 80 ( ° 85 C)

Fig. 90 Fig. 95

10

2 3 0 ). However, with shorter staining dura staining shorter with However, ). a ). We observed a of distribution binding

Fig. Temperature (°C Traptavidin Streptavidin b Pseudomonas aeruginosa

3a Supplementary Boun ) because of the importance of of importance the of because ) Free (%): Free d Supplementary )

100 25 – – + 7.6 25 – + – 9.6 25 1 . We investigated . We investigated – + + – 45 50 Fig. 4.2 50 + 84 60 –

Fig. 13,14 3b FtsK copes + 4.8 60 – + – 99 ). 70

Fig. 3 Fig. 3 , with a with , + 18 70 – b ).

2 c −1 ). ). ). ------

© 2010 Nature America, Inc. All rights reserved. version of the paper at http://www.nature.com/naturemethods/. at paper the of version in the are online available references and any Methods associated M stability. extreme of interactions govern that forces lar may nition also the of aid intermolecu subtle our understanding resonance or point-of-care diagnostics. Traptavidin-biotin recog ple, when used as a anchor for molecular arrays, surface-plasmon many in applications is which dissociation a limitation, for exam in to has streptavidin the replace potential Therefore, traptavidin from gives than higher purification yields yields to streptavidin, and recombinant expression of streptavidin in comparable expressed be recombinantly can Also, traptavidin ( jugates proteins by generation force dissect to curve bration mutants streptavidin weaker used been have nitroavidin assays single-molecule than generation force probe to method simpler motors to obstacle an as used been Streptavidin DNA. previously has the distorting without forces higher of ing is itself deformed is > tweezers 65 pN, the DNA helix point optical at double which even strongly attached DNA streptavidin interaction, ( biotinylated at the 5 equally as strong a roadblock to motors could cooperate in exerting a stronger force.( traptavidin Traptavidin of placement was Increasing the FtsK concentration to 2 observed we little but displacement s, of 2 traptavidin only even afterafter detectable300 s was ( displacement Streptavidin DNA containing a loading site for FtsK and a biotinylated thymidine near the terminusn that was capped with traptavidin or streptavidin (green). FtsK biotinylated bead and an AFM tip coated with streptavidin or traptavidinsingle-molecule were rupturemeasured forces atbetween the a indicated loading rates. Means are andshown streptavidin displacement analysis by AFM: stability compared to streptavidin. ( F Accession protein for 180 s with the indicated concentration of FtsK (analyzed as in ( under each lane. ( traptavidin or without ATP, preventing FtsK activity. The percentage of to freeDNA DNAupon andelectrophoresis, the percentage but boundof DNAstreptavidin displaced byor FtsKtraptavidin for duplicatecauses the assays gel areshift indicatedindicatedof streptavidin with arrows. (SA) orControls traptavidin are (Tr)shown withoutby FtsK streptavidinafter 180 s or was determined by gel domainselectrophoresis, are shownwith DNAin fluorescenceblue and visualized. FtsK does not remain bound e Supplementary igure = 400 and traptavidin, ethods ) Traptavidin is displaced by a high concentration of FtsK. Streptavidin and traptavidin displacement by varying amounts of FtsK analyzed by incubating each Traptavidin binding was more stable for a range of biotin con biotin of for a range more stable was binding Traptavidin with DNA the on pulling by measured FtsK for force stall The Unbinding force (pN) a 100 120 20 40 60 80 3 13,14 0 | Traptavidin has increased mec Loading rate(pN Supplementary 10 . However, only wild-type streptavidin and the weak weak the and streptavidin wild-type only However, .

codes. 3 d ) Streptavidin or traptavidin displacement by FtsK analyzed by incubating each protein with FtsK for the indicated times (analyzed as in

Streptavidin Traptavidin Fig. 4 1 GenBank: GU952124. GenBank: 4 . Traptavidin should enable test displacement ′ terminus rather than at an internal thymidine s n –1 = 562), with best fits and 95% confidence limits indicated by solid and dashed lines, respectively. ( 10 ) ). As FtsK rapidly broke the stable biotin- stable the broke rapidly FtsK As ). 4 in vivo

Fig. γ Fig. in gray. In the presence of ATP, FtsK translocates along the DNA and collides with streptavidin or traptavidin. ( - b binding proteins. 7 a 1

, so traptavidin and the range of of range the and traptavidin so , FtsK should be sufficient to displace 5 ) Traptavidin

could be used to generate a cali a generate to used be could Escherichia coli 3 ), not just one particular ligand. ligand. particular one just not ), e h ), indicating that multiple FtsK multiple that indicating ), FtsK anical

µ d M allowed substantial dis

Streptomyces avidinii Streptomyces Terminal protein: Displaced (%): FtsK and with DNA Boun Free Time (s): d 7 , providing a providing , 1 300 5 0 – S – . Biotinylated DN Fig. 3 0 A 31 1 2 c 43 d ). 3 0 1 6 ). ------60 . 6 0 A 57 0 300 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprintsandpermissions/. R Published online at http://www.nature.com/naturemethods/. text HTML version of the paper at The authors declare competing financial interests: details accompany the full- COM the manuscript. D.J.S. designed research; all analyzed the data; M.H., D.J.S. and C.E.C. wrote C.E.C., M.H., E.C., V.T.M. and C.C. performed the research; M.H., V.T.M. and AUTHOR J.E. Graham and I. Grainge for helpful discussion. the University of Miami (V.T.M.) and Worcester College Oxford (M.H.). We thank Institutes of Health (V.T.M. and C.C.), the National Science Foundation (V.T.M.), Biotechnology and Biological Sciences Research Council (C.E.C.), the US National Funding was provided by the Wellcome Trust (M.H., D.J.S. and E.C.), the A Note: information Supplementary is available on the 72 eprints and permissions information is available online at http://npg.nature. c

k

T no 0 6 0 P Streptavidin or Acids Res. Acids Sci. 1 USA Sci. Acad. Natl. (2002). 3214–3223 (1999). 21–31 (2005). 9949–9959 (2009). Sci. Life Gallizia, A. Gallizia, E. Crozat, P.J.Pease, L.K. Arciszewska, & D.J. V.,Sherratt, Sivanathan, J.E., Graham, R.E. Stenkamp, P.S.& Stayton, L., Klumb, I., Trong, Le S., Freitag, A.D. Ellington, & M. Levy, Cantor,C.R. & Mohr,S.C. Y.,Yu, F.M., Aslan, S.L. Wong, & K.K. Ng, S.C., Wu, B.Vogelstein, K.W.& Kinzler, G., Traverso, H., Yan, D., Dressman, Raney,K.D. & A.J. P.D.,Tackett,Morris, P.Bongrand, & A.M. Benoliel, Touchard,D., A., Pierres, L.A. Sklar, & R. Harris, J., G.P.,Keij, Lopez, T.,Buranda, D.T.Cramb, & R. Heuff, J.L., Swift, M. Akaaboune, & R.I. Hume, D., Sutter, E., Bruneau, Bertozzi,Sletten,&C.R.E.M. M.S. Kulomaa, & V.P.,Hytonen,H.R. O.H., Nordlund, Laitinen, r ETING 0 traptavidin 0.9 w 2 0

, 8507–8512 (2005). 8507–8512 , ledgments 6 CONTRIBUTIONS 300 8.5 , 1157–1166 (1997). 1157–1166 ,

FINANCIAL

6

3 et al. et 3 et al. et , 2992–3017 (2006). 2992–3017 , et al. et 8 , 72–81 (2010). 72–81 , e nature methods

EMBO J. EMBO

Terminal protein: Science Protein Expr. Purif. Expr. Protein

Displaced (%) INTERESTS

1 [FtsK] Boun 00 Free c

, 8817–8822 (2003). 8817–8822 , 2 3 d Terminal protein: ( brief communications 9 07 µ Chem. Biol. Chem. Displaced (%): M) , 1423–1433 (2010). 1423–1433 , h Angew.Engl.Edn.Chem.Int. , 586–590 (2005). 586–590 , t 2 : t Boun p S – Free (%) – Free Proteins : /

/ 78 | d 2 ATP: w A ADVANCE Biophys. J. Biophys.

1 w 65

1 4 w 1 : b , 192–196 (1998). 192–196 , . 5

SA 0.5 19 Methods n ) Cartoon of motor assay showing 67 0 0 – 77 , 979–989 (2008). 979–989 , a t SA 18 6.4 0.2 u , 404–412 (2009). 404–412 , – r Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. e N

14

. 0.1 Tr a 0 0 – 0 90 c ONLINE t

o ± u 23 15 m Tr 1 s.e.m. (streptavidin, , 1396–1410 (2006). 1396–1410 , – r 0 3 0 e / , 149–159 (2001). 149–159 ,

100 100 n M J. Neurosci. J. + T – 1 2 a 1 e r t

t 100 100 u 6.7 PUBLICATION Biophys. J. Biophys. h + –

r c Cytometry o 48 e ) Displacement

d SA m 84 87 6.2 0.5 + s , 6974–6998, e website. t SA 76 81 1.2 0.2 h + Cell. Mol. Cell. α o Nucleic

and

d 3.3 17 Tr 2 0.1 + s 0 0 Proc.

5

/ 3 Protein 8 , .

6.5 20 Tr + 7 2 c 0 β , , ).

|

 © 2010 Nature America, Inc. All rights reserved. per per liter of culture for streptavidin and 5 mg per liter of culture for using OD from dialysis after determined was concentration Protein PBS. against times three (pH 7.8). was dazole dialyzed The eluate imi mM Tris,200 mM 50 NaCl, mM 300 ml 5 with elution for dazole (pH 7.8) and then added to a poly-prep column (Bio-Rad) centrifugation, washed once by with 5 ml of 300 mM NaCl,isolated 50 mM was Tris, resin 30 mM imi the day, next The °C. 4 at overnight Tris, 10 mM imidazole (pH 7.8),mM stirring was added and 50 rotated overnight NaCl, mM and 300 in °C equilibrated 4 (Qiagen), at resin Ni-NTA PBS into dilution rapid by (pH hydrochloride 1.5) (GuHCl). Protein in GuHCl was refolded guanidinium M 6 in dissolved then and 7.8) (pH X-100 Triton was washed three times in 10 ml 100 mM NaCl, 50 mM Tris, 0.5% 27,000 at centrifugation After sonicator. Vibra-Cell Sonics a on amplitude 40% at ice on ml mg 0.8 pH 7.8 for 30 min at 25 °C followed by 9 min EDTA, of pulsed sonication mM 5 10 ml with 300 by mM 750 NaCl,ml rocking culture 50 mM Tris, a of pellet cell the from isolated were bodies Inclusion °C. 37 at h 4 additional an for incubated and IPTG mM 0.5 with induced to OD grown LB into ampicillin, 100-fold diluted a freshly grown colony from of picked traptavidin, or streptavidin of culture overnight Streptavidin described ously previ been have CFP) enhanced to fused H2B histone (human pECFP-H2B and reticulum) endoplasmic the to ( targeted BirA-ER ligase IGF1R. mature of terminus a N the with ing along sequence signal IGF1R 6-amino-acid spacer sequence before the start of sequence encod the after inserted thus into ligated and peptide pcDNA3.1. The acceptor NotI and NheI with digested PCR, extension and IGFA IGFD and IGFC primers ( primers with with fragment second fragment the and first IGFB the fragments: two from V. PCR-amplifying by gift Oxford) of Macaulay, University from was pcDNA3 constructed human IGF1R (a containing kind AP-IGF1R sequencing. DNA by confirmed were mutations The by ( F S52G R53D primer streptavidin the using into mutagenesis (Stratagene) mutation QuikChange by S52G,R53D generated the was Traptavidin introducing pET21a(+). in protein the ing encod gene the with terminus, C the at 6His with streptavidin Plasmid translocating when change to likely is KOPSto and FtsK- that Note 2ve9. and 2iuu 1swe, structures crystal on based PyMOL with constructed in The cartoon Scientific). (DeLano PyMOL using were (1mk5) and biotin and (1swa) displayed with aligned biotin without streptavidin of Structures electrophoresis. during units by surrounded ice to sub of the prevent streptavidin dissociation performed at 200 V with the gel box (X Cell SureLock; Invitrogen) 23 at PBS in dissolved was (Sigma) Avidin General. ONLINE nature methods were with Alexa labeled and traptavidin Streptavidin traptavidin. Supplementary ε 280

METHODS

Biotin (Sigma) was dissolved in DMSO at 100 mM. mM. 100 at DMSO in dissolved was (Sigma) Biotin construction. Supplementary of 34,000 M 34,000 of

and 1 8

Table .

traptavidin −1

Streptavidin in this paper refers to core core to refers paper this in Streptavidin 1 E. coli g ). The fragments were joined by overlap were joined ). fragments The cm

for 15 min, the inclusion body pellet pellet body inclusion the min, 15 for γ Table is shown in the conformation bound bound conformation the in shown is −1 BL21 (DE3) RIPL (Stratagene), was −1 (ref. (ref.

expression lysozyme, 1% Triton X-100 X-100 Triton 1% lysozyme,

1 1 7 ) and its reverse complement. complement. reverse its and ) (GLNDIFEAQKIEWHE) was (GLNDIFEAQKIEWHE) 20). Typical yields were 8 mg mg 8 were yields Typical 20).

and µ M. SDS-PAGE was was SDS-PAGE M.

purification. 600 Figure E. coli E. 1 0.9 at 0.9 37 °C, 3 .

biotin biotin 3 b was An An 280 1 9 ------.

plate-reader. We added 20 was or measured in streptavidin traptavidin PBS on a PHERAstar Biotin–4-fluorescein routine. curve-fitting least-squares linear “LINEST” the using bars, error calculate to (ln( bound) ln(fraction of plot the from determined be to constant ( biotin cold of addition before complex protein-biotin the of radioactivity the ( point time each at supernatant the of radioactivity age aver The BeckmanCoulter). (LS-5000TD; liquid counter a in scintillation counted and cocktail scintillation the to added was at 37 °C. Percentage dissociation was calculated as (signal with with (signal as calculated was dissociation Percentage °C. 37 at started were immediately measurements added, fluorescence and for 1 h at 37 °C. Then, 20 ml mg 0.12 10 1 added We immediately. biotin by re-filled are dissociation so biotin, by open left sites that biotin–4-fluorescein excess using performed was assay The recovers. fluorescence the emission fluorescein of quenching in results protein binding of excess an to fluorescein biotin–4- of LabTech). assay,(BMG In this binding the emission using a PHERAstar plate-reader with 480 nm excitation and 520 nmfluorescein from avidin, streptavidin or was traptavidin measured Biotin–4-fluorescein PBS. in dialysis of rounds three by removed was dye free and pooled were protein labeled containing Fractions column. 1 using ml in packed G-25 volume a (Sigma) Sephadex poly-prep separated 4 °C. h 25 for dye was at Free incubating and mamide) ml mg 1 at dissolved (stock (Invitrogen) ester 555 succinimidyl excess Fluor of Alexa molar a then tenfold NaHCO M 1 of volume 1/10 adding by 555 Fluor Then, Then, 25 centrifugation. by followed °C, 25 at h 1 for 7.8), (pH imidazole of slurry Ni-NTA50% in resin, 300 mM a equilibrated NaCl, 50 mM Tris,with 10 mM incubation by down pulled was complex protein-biotin the point, time each At °C. 37 at incubated and 50 of concentration final a to added was biotin radioactive non To dissociation, PBS. in initiate traptavidin or streptavidin 8,9-[ previously method described that from a modified using determined were traptavidin and streptavidin [ t above. as measured immediately was intensity fluorescence and 8.3, pH at to adjusted to dissociation, pH block further 7.2 1 with M HEPES ice on placed then were pH, low samples at decreased is rescence 100 at 37 °C with with NaCl mM 100 in B4F 30 mM (pH nM citrate sodium 5.0) for 3 h at 25 °C 12 before incubating with incubated was vidin (B4F). to protein was added the binding biotin–4-fluorescein without biotin) × 100. For the signal without quenching, no biotin- biotin − signal without biotin)/(signal without quenching − signal -tests from the triplicate data at the 6 h time point. time h 6 the at data triplicate the from -tests 3 H]biotin For the low pH off-rate assay, 100 nM streptavidin or trapta or streptavidin nM 100 assay, off-rate pH low the For µ a l of PBS to 12 nM biotin–4-fluorescein (Invitrogen) with with (Invitrogen) biotin–4-fluorescein nM 12 to PBS of l 3 − H]biotin (PerkinElmer LAS) for 1 h at 25 °C with 250 nM nM 250 with °C 25 at h 1 for LAS) (PerkinElmer H]biotin x / µ a P )) )) against time. Excel was used for linear regression and l supernatant, containing unbound radioactive biotin, radioactive unbound containing l supernatant,

off-rate −1 values were calculated using two-tailed Student’s Student’s two-tailed using calculated were values BSA in 170 170 in BSA a µ ) enabled the first-order dissociation rate rate dissociation first-order the enabled ) M biotin for 0.5, for 1, M 3,biotin 6 18 h.and As fluo B4F

assay.

off-rate 2 on-rate 1 . dissociates, As the biotin–4-fluorescein µ The dissociation kinetics of biotin from µ µ l l PBS or 20 l l of 10 nM or streptavidin traptavidin l of PBS and incubated the solutions solutions the incubated and PBS of l

assay. assay. The off rate of biotin–4- of rate off The The on rate of B4F for for B4F of rate on The µ l l 1 in mM PBS was biotin 2 2 −1 . WenM 10 incubated doi:10.1038/nmeth.1450 in dry dimethylfor dry in µ 3 (pH 8.3) and and 8.3) (pH M protein in in protein M x ) and and ) µ M - - - - -

© 2010 Nature America, Inc. All rights reserved. in a Thermal Cycler and cooling to 10 °C. A 1.5% agarose gel was Cyclerwas °C. to10 gel cooling agarose Thermal and a 1.5% in A min 3 for temperature indicated the at heating before °C, 25 at min 5 for incubated and 7.5) (pH glutamate potassium mM 20 and acetate magnesium mM DTT, 2 mM 1 Trisacetate, mM 20 were made up to 10 biotinylated DNA in a volume of 4 streptavidin or traptavidin in PBS was incubated with 21 nM mono charge. protein by influenced is position its as weight to molecular according run not does band tetramer the that Note (Bio-Rad). software 4.6 QuantityOne and imager XRS ChemiDoc a using quantified were intensities Band min. for 3 °C 95 at heating before buffer loading SDS with mixed was control positive monomer 100% The gel. an onto polyacrylamide 18% loaded and buffer loading SDS-PAGE 6× with mixed ice on placed immediately then and Cycler indicated the for 3 min at temperature in a DNA Peltier Thermal Bio-Rad Engine heated was PBS in traptavidin or streptavidin Thermostability temperature with increases dramatically it is that known the rate streptavidin of off streptavidin-biotin values previous with comparable Our s.d. the representing letters A ( ( the formula with was calculated quotient The s.d. this of Equilibrium routine. curve-fitting least-squares the linear using “LINEST” bars, error to calculate and regression [ 1/([free of order association rate constant was then determined from the plot [ free calculate to protein, of each addition before present at cpm radioactivity total the in and point time supernatant We the of PerkinElmer). radioactivity 1409; the measured (Wallac counter scintillation liquid [ by centrifugation. Then 25 [ of at time 25 °C dissociation this (in °C 25 at h 1 for 7.8), (pH imidazole Tris, mM mM 10 50 NaCl,mM 300 in Ni-NTA equilibrated of resin, slurry 50% with incubation by down pulled was Protein biotin. nonradioactive nM 1 with traptavidin [ or streptavidin pM 250 incubating by [ (Microsoft). Excel in routine curve-fitting least-squares linear “LINEST” the using calculated were rate on to equal gradient the time, with against biotin–4-fluorescein]) plot ln([free of the to applied was Software) (GraphPad Prism GraphPad signal without B4F) × 50 pM B4F.starting Linear regression using protein − without B4F)/(signal B4F with − without (signal signal approach at 37 °C. The concentration of free B4F was calculated as this using measure to fast too was rate on The °C. 25 at s 6 every 180 to doi:10.1038/nmeth.1450 cm V 6.0 at run 3 3 3 B , H]biotin in PBS at 37 °C. Binding was stopped by addition of 50 H]biotin H]biotin was added to scintillation cocktail and counted in a in counted and cocktail scintillation to added was H]biotin To determine thermostability of biotin conjugate binding, 5.0

± B

and and b ) = ( = ) µ l of 56 pM B4F and measured the fluorescein emission emission fluorescein the measured and B4F pM 56 of l k on C

C on-rate × ([streptavidin or traptavidin]). Error bars for the the for bars Error traptavidin]). or ([streptavidin × are are 3

dissociation

H]biotin]) against time. Excel was used for linear linear for used was Excel time. against H]biotin]) ±

c ), where where ), −1

k 23,26 assay. off µ in TAE for 45 min at 25 °C and ethidium ethidium and °C 25 at min 45 for TAE in 2

assay. 5 L with a final concentration of 100 , (4 × 10 . k on To determine tetramer stability, 3 3 stability, tetramer determine To

and and constant. c The on rate of biotin was determined determined was biotin of rate on The 3 µ = = H]biotin concentration. The second- The concentration. H]biotin l l of supernatant containing unbound −14 3 C H]biotin is negligible H]biotin K × × M) was determined at 25 °C, M) but was determined k d 23,24 on , respectively, with lowercase lowercase with respectively, , µ √

K l l for 30 min at 25 °C. Samples (( and and d . The literature . literature The was calculated from a / A k ) off 2 + ( + for streptavidin are are streptavidin for 27–29 b/B . Samples were were Samples . ) 2 2 K 0 ), in which which in ), ), followed ), followed µ d value for value M biotin, k A off

± / µ µ µ k

a on M M M )/ - .

Cells Cells were washed with PBS/Mg 3 times before live.imaging Cells to 5 cells. adding min before traptavidin to added the fluorescent 50 pre-blocking, For streptavidin. or traptavidin 0.4 and BSA dialyzed 1% with kept thereafter at 4 °C. Cells were incubated for 15 min in PBS-Mg cells were washed 3 times in PBS with 5 mM MgCl day, next The BirA-ER. by biotinylation optimum for overnight 10 with incubated were Cells plate. well 48 a of well with 0.25 instructions manufacturer’s following (Invitrogen) 2000 50 ml U 50 serum, calf fetal 10% with DMEM in Cell derivative pUC18 of containing a lambda phage insert. (Eurofins) ( with the primers Fts1 and the internally biotinylated primer bioFts2 biotinylated DNA was prepared by PCR using Taq DNA polymeraseintensities of the bands for free as and bound DNA. defined The were 439-bp100× themono intensity Percentages of the band software. for free 4.6 DNA divided by QuantityOne the summed using bromide-stained DNA was visualized on a ChemiDoc XRS imager Figure in shown experiment the in concentrations indicated the at and FtsK was added at 0.5 followed by 100 2 with incubated was fragment DNA nM 16 7.5). (pH (DTT) dithiothreitol mM 1 and glutamate potassium mM 20 acetate, magnesium mM 2 acetate, Tris mM 20 in °C 25 at performed was assay displacement FtsK affinity chromatography and heparin affinity chromatography. The in J. (Oxfordof GrahamUniversity). proteinoverexpressedThe was the soluble fragment containing the C-terminal 578 residues including streptavidin or traptavidin ATP,of it would take FtsK loaded at KOPS ~0.5 s to translocate to sites loading (GGGCAGGGGGGCAGGG) KOPS suchpositionedupon that two contained fragment This pJEG41-P1. the plasmid and from (Eurofins) Fts1 bioFts2 primers primer biotinylated using internally polymerase by DNA Taq generated was with PCR fragment DNA monobiotinylated bp 439 a FtsK AFM conditions. identical under analyzed and imaged pared, background- pre were were experiment same images the in samples fluorescence Different corrected. and s 0.5 – 0.1 were times exposure Typical software. 3.6.2 softWoRx using analyzed 600DF50 excitation, emission, Chroma 84100bs polychroic) images were (540D420 collected and 555 Fluor Alexa and dichroic) emission, Chroma excitation, 86002v1 480DF40 ECFP (436DF20 lens. oil-immersion 40× a fluorescent with Core (AppliedPrecision) microscope DeltaVision wide-field a using imaged were adding 1 for the experiment shown in for the experiments shown in 2 mM ATP to start the reaction and incubated the samples for 3 min E. coli E. µ α g ml

culture,

µ

, displacement analysis. β g AP-IGF1R, 0.2 0.2 AP-IGF1R, g

3 and −1 e and purified by ammonium sulfate precipitation,nickelsulfate ammonium by purified and µ and allowed to bind to the DNA for 5 min. Wemin. added 5 for DNA the to bind to allowed and streptomycin. Cells were transfected using Lipofectamine l 0.1% SDS with 200 mM EDTA (pH 8.0). Samples were Supplementary γ

biotinylation domains and a C-terminaldomains a6His and gift tag,kind a was AFM is described in in described is AFM µ M biotin to block free biotin binding sites. PAK4

assay. µ µ M in the experiments shown in M streptavidin or traptavidin for 10 min, 10 for traptavidin or streptavidin M µ For the experiments shown in g BirA-ER and 0.05 0.05 and BirA-ER g 1 3

. and

Table Figure Figure 3 Pseudomonas

µ imaging. M Alexa Fluor 555–conjugated 555–conjugated Fluor Alexa M

1

3c ) from) pJEG41-N1, plasmid a Supplementary d , e . Reactions were stopped by and for the indicated times

COS7 cells were grown cells COS7 aeruginosa µ g H2B-ECFP per per H2B-ECFP g −1 2 nature methods penicillin and and penicillin µ (PBS-Mg) and M biotin was was biotin M FtsK PAK4, a

Note Figure µ M biotin biotin M

Figure addition

1 .

3c , 3 d - - ,

© 2010 Nature America, Inc. All rights reserved. 2 min with a final concentration of 0.1% SDS and 20 0.1% mM of EDTA. 2 a concentration min final with 2.5 mM ATP was added to start the reaction, which1 was stopped after 100 by followed was incubated with 0.5 MgCl mM 10 7.5), Tris(pH mM 25 in described the containing ment Table ( (Sigma-Genosys) bioFts3 primer biotinylated terminally the and Fts1 primer using Taqwith polymerase DNA by PCR end, was generated 226 bp ing sites from the biotinylated monobiotinylated DNA fragment, containing two 8 bp KOPS load displaced were rounded up to zero. percentage free for negative control). − Negativecontrol)/(100 negative values for of free percentage % – free (% × 100 = displaced band for free DNA + intensity of band for bound DNA). Percentage Percentage free = 100 × intensity of band for free DNAbromide–stained / DNA (intensitywas visualized onof a ChemiDoc XRS imager. 1.5% agarose gel in TAE at 6.0 a onV cmloaded and FF) cyanol xylene mM 0.93 blue; bromophenol sucrose,mM M 0.75 (1.2 buffer loading gel DNA,6× mixedwith incubated for a nature methods µ For the experiment shown in M FtsK

1 ) from plasmid pJEG41. pJEG41. plasmid from ) 3 50C 0 . The FtsK displacement assay was performed . at assay 25 was The performed °C FtsK displacement was added and allowed to bind to the DNA for 5 min.

further 15 min to allow FtsK to dissociate from the µ M biotin to block free biotin binding sites. sites. binding biotin free block to biotin M α , µ β M M streptavidin or for traptavidin 15 min, and and γ Supplementary domains of FtsK, was purified as as purified was FtsK, of domains E. coli E. −1 for 45 min at 25 °C. Ethidium 2 FtsK . 5.9 nM DNA nM 5.9 . 50C

Figure , a soluble frag soluble a , Supplementary

4

, a 598-bp fragment fragment - -

28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. (Fuji). software Gauge Image using quantified and scanner a Fuji FLA3000 using for 2 h, in ddH Green (Invitrogen) washed pH 8.3) at 10 V cm mM in Tris,(89 1× gel TBE rose 2 acid, mM EDTA;89 mM boric on a aga 1.5% loading before blue) bromophenol 2.5% glycerol, 50% EDTA, mM 20 7.5), (pH Tris mM (250 10× buffer with loading mixed gel then and min 20 for incubated were Samples 30. 29.

46422–46428 (2001). 46422–46428 (1996). 1319–1324 Sci. Protein (1999). Qureshi, M.H., Yeung, J.C., Wu, S.C. & Wong, S.L. Wong, & S.C. Wu, J.C., Yeung, M.H., Qureshi, M. Wilchek, & S. Ehrlich-Rogozinski, E.A., Bayer, P.S.Stayton, & R.E. Stenkamp, S., Freitag, I., Trong, Le D.E., Hyre, N.M. Green, D.E. Hyre, P.S.Stayton, V.& Chu, L.A., Klumb, P.S.Stayton, & A. Chilkoti, H.J. Gruber, & H. Falk, G., Kada, Cantor,C.R. & T.Sano, A.Y.Ting, & M. Howarth, M. Howarth, P.J.Schatz, & E. Kovaleva, D., Beckett, Lowe, J. Lowe, S.L. Wong, & M.H. Qureshi, et al. et et al. et

9 Methods Enzymol. Methods et al. et , 878–885 (2000). 878–885 ,

Mol. Cell Mol.

Protein Sci. Protein −1

Nat. Methods Nat. for 2 h at 25 °C. Gels were stained with SYBR

Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. 31 Nat. Protocols Nat. , 498–509 (2008). 498–509 ,

J. Am. Chem. Soc. Chem. Am. J. Protein Expr. Purif. Expr. Protein 1

5 1 , 459–467 (2006). 459–467 ,

84 5 Biochim. Biophys. Acta Biophys. Biochim. , 397–399 (2008). 397–399 , , 51–67 (1990). 51–67 , Biochemistry

3 Protein Sci. Protein , 534–545 (2008). 534–545 ,

11 2 2 doi:10.1038/nmeth.1450 O O for 30 min, imaged 5

Electrophoresis 7 3 87 , 409–415 (2002). 409–415 , J. Biol. Chem. Biol. J. , 10622–10628 (1995). 10622–10628 , 7

8 , 7657–7663 (1998). 7657–7663 , , 142–146 (1990). 142–146 , , 921–929 (1999). 921–929 ,

1 4 2 7 , 33–43 ,

1

2 7 76 ,

, -