ITC experiments
were performed with a Microcal VP-ITC microcalorimeter (Northampton,
MA). Data were analyzed using nonlinear least-squares curve fitting
in Origin7.0 (OriginLab Corp.).
NEO1A in the reaction cell and
neomycin-B in the syringe were prepared in the same buffer for each
experiment as previously described.41 (link) All
solutions were degassed at room temperature, and following thermal
equilibrium at 25 °C and an initial 60 s delay, 30 serial injections
of neomycin-B were added at an interval of 300 s into the stirred
sample cell (1.4 mL) containing the NEO1A variant at a stirring rate
of 310 rpm at 25 °C. The heat associated with each titration
peak was integrated and plotted against the respective molar ratio.
Control experiments were performed to correct for the heats of dilution
from the titrants by making identical injections of the titrant solution
into a cell containing only the respective buffer, and these values
were subtracted from the titration of the titrant solution into the
reaction cell. Data were analyzed using the standard one-binding site
model fitting (nonlinear least-squares curve) in Origin7.0 (OriginLab
Corp., Northampton, MA).
were performed with a Microcal VP-ITC microcalorimeter (Northampton,
MA). Data were analyzed using nonlinear least-squares curve fitting
in Origin7.0 (OriginLab Corp.).
NEO1A in the reaction cell and
neomycin-B in the syringe were prepared in the same buffer for each
experiment as previously described.41 (link) All
solutions were degassed at room temperature, and following thermal
equilibrium at 25 °C and an initial 60 s delay, 30 serial injections
of neomycin-B were added at an interval of 300 s into the stirred
sample cell (1.4 mL) containing the NEO1A variant at a stirring rate
of 310 rpm at 25 °C. The heat associated with each titration
peak was integrated and plotted against the respective molar ratio.
Control experiments were performed to correct for the heats of dilution
from the titrants by making identical injections of the titrant solution
into a cell containing only the respective buffer, and these values
were subtracted from the titration of the titrant solution into the
reaction cell. Data were analyzed using the standard one-binding site
model fitting (nonlinear least-squares curve) in Origin7.0 (OriginLab
Corp., Northampton, MA).
