Construction and characterization of a temperature-controlled fluid cell for single-molecule studies with Magnetic Tweezers

Abstract

An important class of enzymes that is object of single-molecule biophysics studies is the one formed by proteins called helicases. Helicases are molecular motors that move along double-stranded DNA unwinding the two strands using the energy of ATP. These motor proteins can have different structural compositions that influence their function, but all of them have the property that their activity depends on the temperature of the surrounding medium. Our goal is to study how the activity of the helicase-nuclease complex AddAB is influenced by temperature, using a Magnetic Tweezers (MT) setup. With the aim of controlling the temperature inside the sample cell with a precision of 0.1 °C, a modified version of the current MT sample cell has been constructed and characterized. Briefly, the sample cell consists of two glass cover slides sealed by two layers of Parafilm. This assembly is coupled to liquid inlets and outlets by a metal baseplate and a plastic sample holder. The channel created between the two glass layers is accessed through two holes made in the upper cover glass and enables flushing buffer inside the fluid cell to study the activity of biological samples. In order to control the temperature of the system two thin-foil resistive heaters have been attached to the bottom part of the baseplate, and using a high-precision sensor the temperature of the baseplate has been monitored. The setup is controlled by customized LabVIEW software that contains a PID feedback, through which the voltage applied to the heaters is regulated and the heating process is monitored. The results presented in this thesis correspond to calibration measurements and finite-element simulations. We have found that both in-silico and experimental measurements are in good agreement.