Protein aliquots were thawed
and solubilized at 50 °C and then diluted to 6 μM in buffer
to adjust to a final salt concentration of 150 mM in 20 mM, Tris-HCl,
pH 8.5. SYNZIP-tagged constructs were similarly adjusted to 10 μM
in 150 mM NaCl, 20 mM HEPES, pH 6.8. The protein mixture, 60 μL
in volume, was added to quartz microcuvettes (10 mm path length) (Starna
Cells, Inc. Atascadero, CA). Cuvettes were inserted into a Cary 3500
UV–vis spectrophotometer controlled by an Agilent multizone
Peltier temperature controller (Agilent Technologies; Santa Clara,
CA). To test kinetics of rapamycin-induced dimerization and phase
separation of FRB and FKBP-tagged RGG constructs, rapamycin (Sigma-Aldrich;
St. Louis, MO) was spiked into the protein mixtures to a final concentration
of 10 μM and absorbance at 600 nm was measured over time. For
mapping the temperature-dependent phase separation, protein mixtures
were applied to quartz cuvettes preincubated at 50 °C. Cuvettes
were then inserted into the preheated spectrophotometer, set to 50
°C, and samples were cooled to 5 °C at a rate of 1 °C/min
while measuring the absorbance at 600 nm.
and solubilized at 50 °C and then diluted to 6 μM in buffer
to adjust to a final salt concentration of 150 mM in 20 mM, Tris-HCl,
pH 8.5. SYNZIP-tagged constructs were similarly adjusted to 10 μM
in 150 mM NaCl, 20 mM HEPES, pH 6.8. The protein mixture, 60 μL
in volume, was added to quartz microcuvettes (10 mm path length) (Starna
Cells, Inc. Atascadero, CA). Cuvettes were inserted into a Cary 3500
UV–vis spectrophotometer controlled by an Agilent multizone
Peltier temperature controller (Agilent Technologies; Santa Clara,
CA). To test kinetics of rapamycin-induced dimerization and phase
separation of FRB and FKBP-tagged RGG constructs, rapamycin (Sigma-Aldrich;
St. Louis, MO) was spiked into the protein mixtures to a final concentration
of 10 μM and absorbance at 600 nm was measured over time. For
mapping the temperature-dependent phase separation, protein mixtures
were applied to quartz cuvettes preincubated at 50 °C. Cuvettes
were then inserted into the preheated spectrophotometer, set to 50
°C, and samples were cooled to 5 °C at a rate of 1 °C/min
while measuring the absorbance at 600 nm.