The remarkably strong interaction between the small molecule biotin and the proteins streptavidin or (neutr)avidin is widely exploited in biological research1
. Biotin-binding proteins have been isolated from a wide range of species but streptavidin shows the most stable binding to biotin-conjugates1
. Streptavidin is used in imaging, protein purification and nano-assembly, while also showing success in cancer clinical trials. Streptavidin and biotin have low non-specific binding, while biotinylation generally does not disrupt biomolecule function. Alternative targeting methods, such as HaloTag or SNAP-tag, form irreversible covalent bonds to their ligand and are valuable for cellular labeling2
. However, these domains do not have the resistance of streptavidin to temperature, pH, or denaturant, and so even though pre-bound ligand will remain attached under these harsh conditions, these covalent-binding proteins may unfold, aggregate and promote non-specific binding. Also, unlike SNAP-tag and HaloTag ligands, biotin can be precisely targeted to proteins in vitro
, on cells and in living animals using biotin ligase1
. An additional advantage is that a large variety of streptavidin- and biotin-conjugates are commercially available.
Despite its stable binding, the perception that the streptavidin-biotin interaction is essentially irreversible is far from correct. For example, in imaging, low endosomal pH led to dissociation of streptavidin being detected in 2 h, whereas the receptor of interest had a lifetime of ~4 days3
. Also, nanoparticle attachment can cause a surprising decrease in streptavidin-biotin stability4
; the Kd
for a biotinylated peptide increased approximately a million-fold when streptavidin was attached to beads5
. In the presence of shear-forces lower than a blood-capillary, streptavidin-coated beads do not attach to a biotinylated surface but instead roll across, with arrests of 20 ms to tens of seconds6
. In addition, streptavidin cannot prevent the translocation of molecular motors such as helicases, RNA polymerase or DNA polymerase along DNA7
. Streptavidin is used at high temperatures, such as for PCR, BEAMing and 454 DNA sequencing, but DNA has to be bis-biotinylated to reduce dissociation8
A streptavidin mutant containing a cysteine formed a disulfide with a thiol-linked biotin-conjugate, giving controlled reversibility9
, but this mutant only enhances binding to certain biotin-conjugates and in systems unaffected by changing redox, precluding use on cells. We therefore endeavored to engineer a streptavidin mutant that would bind more stably to any biotin-conjugate.
In a highly optimized system, almost any change reduces performance. Over two hundred mutants of streptavidin have been published but none have had improved biotin binding stability1
. Streptavidin libraries have been screened for various properties by phage-display and in vitro
compartmentalization, yielding, for example, a streptavidin variant with improved desthiobiotin binding, but no pair with as strong binding as wild-type streptavidin-biotin has been identified10, 11
. Based upon this literature, we avoided mutations near the ureido or thiophene rings of biotin, which invariably impair binding1
, and explored numerous mutations adjacent to the biotin carboxyl and in the L3/4 loop1, 10, 11
. We randomized promising residues and evaluated purified proteins according to biotin-4-fluorescein off-rate, finding the lowest off-rate for the S52G R53D mutant of streptavidin (), which we termed traptavidin. We hypothesize that the mutations in traptavidin reduce flexibility of the L3/4 loop (residues 45–50, ). Upon biotin binding, this loop becomes ordered and closes over the biotin-binding pocket12
. A more ordered loop may reduce the entropic cost of biotin binding and inhibit dissociation, while decreasing the on-rate and enhancing thermostability.
Traptavidin exhibits a slower off-rate from biotin-conjugates
The off-rate for free biotin at 37 °C and pH 7.4 was > 10-fold lower for traptavidin than streptavidin (4.2 ± 0.5 × 10−6
for traptavidin, 6.8 ± 0.3 × 10−5
for streptavidin, ). Since a substantial part of (strept)avidin’s binding energy comes from interaction with the carboxyl group of biotin1
, it is important to establish how derivation at the carboxyl group changes binding strength. Traptavidin also showed a dramatically reduced off-rate to biotin-conjugates (, P
= 0.0008). After the ~2% dissociation at the initial time-point, there was little dissociation from traptavidin over the subsequent 12 h. In contrast, streptavidin dissociated steadily, while avidin dissociated even faster than streptavidin1
. At pH 5, traptavidin dissociation was faster than at pH 7.4 but was still significantly slower than streptavidin (P
= 0.001) (). The on-rate of traptavidin for biotin-4-fluorescein was reduced two-fold, from 2.0 ± 0.1 × 107
for streptavidin to 1.0 ± 0.03 × 107
for traptavidin (P
= 0.004), while the on-rate of traptavidin for 3
H-biotin was also reduced (Supplementary Fig. 1
). The slower on-rate of traptavidin means that longer incubations are required to reach equilibrium.
Streptavidin is often used at high temperature. Traptavidin had increased thermostability compared to streptavidin before splitting into monomers; the midpoint of transition was ~10 °C higher (). We also assessed biotin-conjugate binding stability at elevated temperatures (). At 70 °C there was complete dissociation from streptavidin but most ligand was still bound to traptavidin.
Traptavidin shows increased thermostability
Imaging of cell-surface proteins using biotin ligase and streptavidin is rapid and sensitive and the target-protein needs only to be modified with a 15 amino-acid tag. The altered charge of traptavidin may affect non-specific cellular binding, as seen for avidin compared to lower pI mutants1
. We investigated whether traptavidin showed similar specificity to streptavidin on mammalian cells. We fused the type 1 insulin-like growth factor receptor (IGF1R) to the acceptor peptide (AP-IGF1R), biotinylated the AP with co-expressed biotin ligase (BirA-ER), and detected biotinylated AP-IGF1R with fluorescently-labeled traptavidin or streptavidin (Supplementary Fig. 2
). Traptavidin showed high specificity for imaging, with a strong signal on cells expressing AP-IGF1R and BirA-ER and minimal binding when traptavidin was pre-blocked with biotin. Staining with traptavidin and streptavidin was comparable (Supplementary Fig. 2
). However, with shorter staining times, the cell staining was more intense with streptavidin (data not shown), consistent with the slower on-rate of traptavidin.
The relationship between binding stability over time versus resistance to force is complex: force changes the height and landscape of the activation-energy for dissociation. We probed the mechanical strength of traptavidin at the single-molecule level by atomic force microscopy (AFM). Traptavidin had greater mechanical binding stability than streptavidin over a range of loading-rates (P
< 0.0001) (). We observed a distribution of binding strengths (Supplementary Fig. 3a
) because of the significance of thermal-fluctuations to traversing the activation-barrier. From the relationship between loading-rate and rupture-force, we were able to estimate the difference in dissociation-rate between streptavidin and traptavidin at a given force (Supplementary Fig. 3b
Traptavidin shows increased mechanical stability
We applied traptavidin to study FtsK, one of the fastest molecular motor proteins, translocating along DNA at 5 kb/s13, 14
. Before bacteria divide, FtsK runs along DNA until encountering XerC and XerD; then FtsK activates site-specific recombination by XerC/D, separating chromosome dimers and ensuring faithful partition of one chromosome to each daughter cell. DNA in vivo
is bound to many proteins, including repressors, transcription factors, RNA polymerases and DNA-bending architectural proteins. To study how Pseudomonas aeruginosa
FtsK copes with obstacles to its journey, we used a short DNA substrate containing KOPS, an 8 bp sequence that loads FtsK directionally13, 14
, with a biotinylated nucleotide near the end, so that FtsK would load on to the DNA in a defined orientation and then translocate until encountering streptavidin or traptavidin ().
Despite the strength of the streptavidin-biotin interaction, FtsK displaced the majority of streptavidin from the DNA within 3 min, whereas traptavidin resisted displacement (P
= 0.003) (). Streptavidin displacement was detectable after only 2 s, but we observed little displacement of traptavidin even after 300 s (). Increasing the FtsK concentration to 2 µM allowed substantial displacement of traptavidin (), indicating that multiple FtsK motors could cooperate in exerting a stronger force. Traptavidin was equally a strong roadblock to Escherichia coli
FtsK and with DNA biotinylated at the 5’-terminus rather than at an internal thymidine (Supplementary Fig. 4
). Since FtsK rapidly broke the stable biotin-streptavidin interaction, FtsK in vivo
should be sufficient to displace even strongly attached DNA-binding proteins.
The stall-force for FtsK measured by pulling on the DNA with optical tweezers was > 65 pN, at which point the DNA double-helix itself is deformed14
. Traptavidin displacement should enable testing of higher forces without distorting the DNA. Streptavidin has previously been used as an obstacle to motors7
, providing a simpler method to probe force-generation than single-molecule assays13, 14
. However, only wild-type streptavidin and the weak nitroavidin have been used7
, so traptavidin and the range of weaker streptavidin mutants1
could act as a calibration curve to dissect force-generation by proteins15
Traptavidin binding was more stable for a range of biotin-conjugates (Supplementary Fig. 5
), not just one particular ligand. Also, traptavidin can be recombinantly expressed in comparable yields to streptavidin and recombinant expression of streptavidin gives yields higher than purification from Streptomyces avidinii16
. Therefore traptavidin has the potential to replace streptavidin in many applications where dissociation is a limitation, for example when used as a molecular anchor for arrays, surface-plasmon-resonance, or point-of-care diagnostics. Traptavidin-biotin recognition may also aid our understanding of the subtle intermolecular forces that govern interactions of extreme stability.