215
the secondary amplifier HCV
TVMV
-AI-SH3
Pep
has been cleaved
and activated to accurately determine the strength of binding
interactions.
1. Optional: Preincubate 50 μL of 10 μM FKBP12-TVMV
Thr
-AI
with 1 μL of 1 U/μL thrombin to cleave off the AI domain.
For reduced signal-to-noise ratios, the AI domain can remain
attached (see Note 11).
2. Incubate 50 μL of 400 pM FKBP12-TVMV
Thr
-AI together
with 50 μL of 20 μM HCV substrate (giving rise to a final concentration of the HCV substrate 5 μM) and 50 μL of varying
concentrations of rapamycin. With an anticipated K D in the
sub-nM range for the FKBP12-rapamycin interaction, the preferred concentration of the primary FKBP12-TVMV
Thr
-AI
sensor in the final assay is 100 pM to prevent titrating the
amount of FKBP12-TVMV
Thr
-AI and not the strength of the
FKBP12- rapamycin binding interaction. Similarly, to reliably
determine its K D for FKBP12, rapamycin is applied between
0.01 and 10 nM.
3. Initiate the reaction with 50 μL of 800 nM HCV
TVMV
-AIFRB. The preferred concentration of the secondary amplifier
HCV
TVMV
-AI-FRB in the final assay is 200 nM, but can be
optimized empirically depending on the signal-to-noise ratio
and the desired response rate.
4. Monitor the reaction progress using a fluorescence 96-wellplate reader by detecting the release of 7-methoxycoumarinyl4-acetyl from the quenched HCV peptide substrate with ʎ ex/em
of 330 and 405 nm.
5. For the quantitative analysis, extract initial rates from the change
in fluorescence (after subtracting the background fluorescence in
the no enzyme control at each substrate concentration) and plot
against the substrate concentration. Crucially, for cascaded systems, the initial rates need to be extracted from the exponential
phase before all of the secondary amplifier HCV
TVMV
-AI- FRB has
been cleaved and activated to accurately determine the strength
of FKBP12-rapamycin-binding interaction.
4 Notes
1. Depending on specific protease switch being assayed, the NaCl
concentration in the protease assay buffer needs to be optimized and tailored toward each application. For instance,
SH3
Dom
-TVMV-FN3-PDZ-AI allosteric receptors display
higher induction ratios at 1 M NaCl compared to 100 mM
NaCl. In contrast, the activity of HCV-based switches is
3.5.2 Proximity Sensors
for Protein Interactions
Engineering Synthetic Protease Switches
the secondary amplifier HCV
TVMV
-AI-SH3
Pep
has been cleaved
and activated to accurately determine the strength of binding
interactions.
1. Optional: Preincubate 50 μL of 10 μM FKBP12-TVMV
Thr
-AI
with 1 μL of 1 U/μL thrombin to cleave off the AI domain.
For reduced signal-to-noise ratios, the AI domain can remain
attached (see Note 11).
2. Incubate 50 μL of 400 pM FKBP12-TVMV
Thr
-AI together
with 50 μL of 20 μM HCV substrate (giving rise to a final concentration of the HCV substrate 5 μM) and 50 μL of varying
concentrations of rapamycin. With an anticipated K D in the
sub-nM range for the FKBP12-rapamycin interaction, the preferred concentration of the primary FKBP12-TVMV
Thr
-AI
sensor in the final assay is 100 pM to prevent titrating the
amount of FKBP12-TVMV
Thr
-AI and not the strength of the
FKBP12- rapamycin binding interaction. Similarly, to reliably
determine its K D for FKBP12, rapamycin is applied between
0.01 and 10 nM.
3. Initiate the reaction with 50 μL of 800 nM HCV
TVMV
-AIFRB. The preferred concentration of the secondary amplifier
HCV
TVMV
-AI-FRB in the final assay is 200 nM, but can be
optimized empirically depending on the signal-to-noise ratio
and the desired response rate.
4. Monitor the reaction progress using a fluorescence 96-wellplate reader by detecting the release of 7-methoxycoumarinyl4-acetyl from the quenched HCV peptide substrate with ʎ ex/em
of 330 and 405 nm.
5. For the quantitative analysis, extract initial rates from the change
in fluorescence (after subtracting the background fluorescence in
the no enzyme control at each substrate concentration) and plot
against the substrate concentration. Crucially, for cascaded systems, the initial rates need to be extracted from the exponential
phase before all of the secondary amplifier HCV
TVMV
-AI- FRB has
been cleaved and activated to accurately determine the strength
of FKBP12-rapamycin-binding interaction.
4 Notes
1. Depending on specific protease switch being assayed, the NaCl
concentration in the protease assay buffer needs to be optimized and tailored toward each application. For instance,
SH3
Dom
-TVMV-FN3-PDZ-AI allosteric receptors display
higher induction ratios at 1 M NaCl compared to 100 mM
NaCl. In contrast, the activity of HCV-based switches is
3.5.2 Proximity Sensors
for Protein Interactions
Engineering Synthetic Protease Switches