Table 1.
Mass measurements.
Fig 1.
S. dysgalactiae CnaB domain analysis.
(a) Graphical view of domains in S. dysgalactiae fibronectin-binding protein, accession code: CAA80122.1. This was not drawn to scale. (b) Sequence alignment of S. pyogenes and S. dysgalaticae CnaB domains using Clustal Omega [21] and ESPript 3.0 [22]. Structural beta-sheet elements (β1- β10) of the S. pyogenes protein are also indicated based on resolved S. pyogenes domain (PDB ID: 2X5P [23]). Residues involved in isopeptide formation are indicated with stars. The S. dysgalactiae CnaB domain used for alignment is located at amino acids 828–950 of the S. dysgalactiae fibronectin-binding protein (1117 amino acids long).
Fig 2.
Optimization of S. dysgalactiae Catcher.
(a) Alignment of I-TASSER homology model of S. dysgalactiae CnaB domain (green) and SpyTag-SpyCatcher complex (pink, 4MLI [24]). Depiction of structure was performed using PyMOL [25]. Inset depicts residues at position 119 (Tag) and 34 (Catcher) for SpyCatcher-SpyTag (pink) and SdyCatcher-SdyTag (green). (b) Schematic diagram of the Catcher variants. Amino acid sequences of the constructs can be found in S1 Table. Residue and beta sheet numbering as follows from Fig 1B. *SpyCatcher sequence as follows from Zakeri et al [10]. (c) Yield (%) of Catcher: SdyTag-EGFP product, with respect to the limiting Catcher substrate, from in vitro reaction of Catcher with 1.3 equivalent SdyTag-EGFP for 40 minutes at 25°C, pH 7. Averages of triplicate measurements are shown and their standard deviations are represented by error bars. Yield % are labelled. (d) Covalent ligation of SdyTag-EGFP with Catchers for 80 minutes at 25°C, pH 7. Lane 1: Novex Sharp ladder (Thermo Fisher Scientific), lane 2: SdyTag-EGFP alone, lane 3: SdyCatcherDANG short alone, lane 4: SdyTag-EGFP incubated with SdyCatcherDANG short, lane 5: SdyTag-EGFP incubated with SpyCatcher, lane 6: SdyTag(Asp117Ala)-EGFP incubated with SdyCatcherDANG short.
Fig 3.
(a) Alignment of Tags. Hydrophobic, neutral, negatively-charged and positively-charged residues are colored as green, black, red and blue respectively. The residue numbering follows that of Fig 1B. The reactive Asp117 is indicated with a star. (b, c) pH dependence of SpyTag and SdyTag. (b) Incubation of SpyTag-EGFP with SpyCatcher (left) and SdyCatcherDANG short (right) at pH 5.2, 6.3, 6.6, 6.8, 7, 8 and 9 after 10 minute at 25°C. (c) Incubation of SdyTag-EGFP with SpyCatcher (left) and SdyCatcherDANG short (right) at pH 5.2, 6.3, 6.6, 6.8, 7, 8 and 9 after 30 minute at 25°C.
Table 2.
Reaction rate constants between different ligation partners.
Fig 4.
Kinetic control for directed protein assembly.
Predicted preferences, based on observed reaction rates of SpyCatcher with (a) SpyTag-EGFP-SdyTag construct (preference for SpyTag versus SdyTag) compared to (b) SpyTag-EGFP-SpyTag construct (no preference for either SpyTag). Predicted preferences for SdyTag-EGFP are shown in (c) SpyCatcher-SdyCatcherDANG short (preference for SdyCatcher) and (d) SpyCatcher-SpyCatcher (minimal reactivity expected). Bold arrows represent fast reaction compared to the dotted arrows which represent slow or minimal reactions. (e) Reactions of the dual-Tags constructs. Reaction of SpyTag-EGFP-SdyTag with SpyCatcher at 5 minutes (lane 1) and 30 minutes (lane 2). At 5 minutes, aliquots were removed and incubated with additional SpyCatcher (lane 3) or additional SdyCatcherDANG short (lane 4) for a further 25 minutes. Reaction of SpyTag-EGFP-SpyTag with SpyCatcher at 5 minutes (lane 5) and 30 minutes (lane 6). (f) Reactions of the dual-Catcher constructs. Lane 1–4 are standalone purified SpyCatcher- SdyCatcherDANG short, SdyTag-EGFP, SpyTag-EGFP, SpyCatcher-SpyCatcher respectively. Incubation of SpyCatcher- SdyCatcherDANG short with excess SdyTag-EGFP for 30 and 40 minutes (lanes 5 and 6). An aliquot of the reaction after 30 minutes was removed and incubated with excess SpyTag-EGFP for further 10 minutes (lane 7). Reaction of SpyCatcher-SpyCatcher with excess SdyTag-EGFP for 40 minutes (lane 8). Excess SpyTag-EGFP was incubated with an aliquot of the latter reaction, after 30 minutes, for a further 10 minutes (lane 9). Astericks indicate the reactions which additional substrates were incubated in the reactions. Novex Sharp pre-stained ladder was used in both gels.
Fig 5.
Construction and polymerization of circular proteins.
(a) Schematics for in vivo and in vitro ligations of triSdyTag constructs. (b) Western blot showing E.coli lysates for the production of triSdyTag with 100 μM IPTG, 16°C (lane 2) and 37°C (lane 1). (c) Characterization of triSdyTag. Lane 1: Purified triSdyTag, lane 2: purified triSdyTag after TEV digestion overnight, lanes 3–7: reaction of SdyCatcherDANG short and triSdyTag at 1, 5, 20, 90 minutes and overnight at 25°C, pH 7 and lane 8: Novex Sharp ladder. (d) Presence of free SpyTag was examined by reaction of SpyCatcher with either SpyTag-EGFP (lane 1), SdyTag-EGFP (lane 2) or circularized triSdyTag (lane 3) at 5 minutes, pH 7, 25°C. Only SpyTag-EGFP (lane 1) has a product (SpyTag-EGFP:SpyCatcher) with SpyCatcher under these conditions. (e) Polymerization of tri-SdyTag. Lanes 1–3: Reaction of bi-SdyCatcher with triSdyTag at 30 μM in 2:1, 1:1, 1:4 Catcher: Tag molar ratios, lane 4: Novex Sharp ladder and lanes 5–8: reaction of bi-SdyCatcher with triSdyTag at 10 μM in 4:1, 2:1, 1:1, 1:4 Catcher: Tag molar ratios. The reactions were left overnight at 25°C, pH 7.