Figure 1.
Schematic illustration of IEkMCC ligands tethered onto a plastic surface with PEG polymer linkers.
IEkMCC proteins with free c-terminal cysteines were first conjugated with heterobifunctional PEG linkers Mal-PEG-Bio through interactions between the sulfhydryl group and the maleimide group. Conjugates with biotin at the free ends of the polymer were then tethered to a plastic surface coated with streptavidin.
Figure 2.
Characterization and separation of PEG polymer linkers and IEkMCC-PEG conjugates.
(A) Compiled elution curves of nine PEG polymer linkers from a Superdex 200 10/300 GL gel filtration column. The polymers were detected through the weak UV absorption of the biotin group using a 245 nm UV detector. (B) Separation of the IEkMCC and PEG polymer reaction products. The reaction products were loaded on a Superdex 200 10/300 GL gel filtration column to separate IEkMCC-PEG conjugates, unreacted IEkMCC, and unreacted PEG polymers. The reaction products of PEG 15000, PEG 30000 and PEG 60000 were first purified with an IEk-binding affinity column to eliminate unreacted PEG polymers. The dotted vertical line indicates the elution volume of IEkMCC protein. The late elution peaks of unreacted polymers can be seen for PEGs ranging from PEG 88 to PEG 5000. In reaction with PEG 7500, unreacted IEkMCC and unreacted polymer formed a single peak that was eluted at a position between unconjugated IEkMCC and pure PEG 7500.
Table 1.
PEG linkers and properties.
Figure 3.
FRET between streptavidin on plastic plates and IEkMCC tethered with PEG polymers.
(A) Measured FRET efficiencies of IEkMCC tethered with six different PEG polymers. The intensity of DyLight 549 was captured before and after DyLight 649 was photobleached. The measured FRET efficiency () was calculated using the intensity of DyLight 549 before (
) and after (
) DyLight 649 photobleaching (
). The averaged values of two measurements were plotted with standard deviations. (B) After normalization, the measured FRET efficiencies match those calculated based on the Flory radius (
) of the PEG polymers. The
of the PEG polymer of
subunits and unit length
was calculated using
, where
is 0.28 nm [32]. Theoretical FRET efficiency (
) was calculated using the equation
, where the Förster distance (
) of the DyLight 549-DyLight 649 donor-acceptor pair is 5 nm and the distance between the pMHC ligand and streptavidin
is
of the PEG polymer plus the pMHC radius of 2 nm. The FRET efficiencies were normalized by dividing the FRET efficiencies by the FRET efficiency of PEG 88.
Figure 4.
T cell activation by IEkMCC tethered with PEG polymers of different lengths.
(A) T cell IL2 production in response to IEkMCC-PEG ligands of varying coating densities after 6 hours of stimulation. Data are representative of three independent experiments. The percent of T cells producing IL2 was determined by intracellular staining and flow cytometry. Three experiments using T cells from three different mice were performed (see Fig. S8 for flow cytometry plots). The percent of T cells producing IL2 was normalized to the highest value in each experiment. The data points are averages of the normalized values with standard errors of the means. (B) The rate of T cell response to IEkMCC ligands tethered with PEG polymers of different lengths. T cell IL2 production in response to stimulation on 96 well plates coated with 110 pM IEkMC-PEG ligands. T cells were harvested every hour for 6 hours and levels of IL2 expression were assayed by flow cytometry. Three experiments using T cells from three different mice were performed (see Fig. S9 for flow cytometry plots). The percent of T cells producing IL2 was normalized to the highest value in each experiment. The data points are averages of the normalized values with standard errors of the means. (C) The rates of T cell IL2 responses to IEkMCC ligands tethered with PEG polymers were extracted from the slope of linear fitting curves in Fig. 4B and plotted against the Flory radius of the polymers. The linear regressions and equations for deriving the rates are shown in Fig. S7.
Figure 5.
The impact of polymer length on pMHC-TCR binding kinetics based on CG-MD simulation.
The on-rates and off-rates derived from the simulation for the PEG 4000, PEG 10000 and PEG 20000 were scaled against the data for the PEG 4000 and plotted as a function of the polymer Flory radius. To display the relationship between binding rates and TCR triggering efficiency, the experimentally determined IL2 expression rates for PEG 3500, PEG 5000, PEG 7500, PEG 150000 and PEG 30000 shown in Fig. 4A were scaled against PEG 3500 and plotted as a function of the polymer Flory radius.