Facilities of the Biochemistry Laboratory in the Faculty of Chemical and Pharmaceutical Sciences, University of Chile: The work of this project will take place mainly at the Biochemistry Laboratory. The Laboratory of Biochemistry has all the basic facilities for the proposed studies (glass-ware, chemicals, pH- meter, refrigerators, cold chamber, stirrers, and thermo-regulated baths, shaker-coupled incubator). In this laboratory also work the research groups headed by Dr. Maria Antonieta Valenzuela and Dr. Mauricio Baez with whom we share equipment. It is equipped with ultracentrifuges, refrigerated centrifuge Sorvall, Eppendorf table-top centrifuge, micro-centrifuge, densitometer, orbital stirrers, electro-transference equipment semi-dry and humid, gel driers, spectrophotometers, termocyclers (PCR instruments), electrophoresis equipment, Pulse-field electrophoresis equipment, Table top autoclaves, animals cells stove; a flake ice maker, an ultrafreezer at –80° and several refrigerators and freezers at –20°, a Beckman ultracentrifuge Type 90, SpeedVac Concentrator Savant, IPGphor Amersham (isoelectrofocusing), a microplate reader, Shakers and all what is needed for bacterial culture and growing.
Single molecule mechanical unfolding can be achieved by means of the analytical “optical tweezers" manipulation. Briefly, this technique uses the light to create an optical trap which can manipulate molecules (Ashkin et al., 1970; Ashkin et al., 1986). This is a direct experimental approach that we will use to study the translocation rate and force at single molecule level. Optical tweezers work through the trapping of polystyrene beads by a light beam (Fig. 4A). The catching mechanism is due to the balance of two types of optical forces: scattering forces, which push the object in the direction of the light beam, and gradient forces, which attract the particle to the focal point. When the gradient forces exceed the scattering forces, particle is stably trapped. Generally, a near-infrared laser beam is used, whose wavelength is not absorbed and thus remains innocuous to most biological materials. Such near-infrared laser beam is tightly focused by a high-numerical-aperture microscope objective lens to create the large spatial gradient in light intensity necessary to form a stable trap. To a first approximation, the trap behaves as a Hookean spring; forces can be generated on an object when it is displaced from the center of the trap and the force can be calculated from a product of the spring constant of the trap k, and the object displacement, Δx.
Figure 4. Experimental setup. System to study the forces involved in the conformational changes of BiP during translocation . A. Optical tweezers work through the trapping of polystyrene beads by a light beam. BiP protein will be attached to little balls of polystyrene by means of DNA handles. These DNA handles will be derivatized in its 5’ ends with digoxigenin and biotin, to bind the respective beads (antidigoxigenin coated beads and streptavidin coated beads, respectively). The streptavidin bead will be attached to a micropipette by suction and has a diameter of 2.1 μm. This system will be inside a laminar flow camera, which allows buffer change (with and without substrate). B. The prepro alpha factor (in black) is the protein substrate to be translocated by the translocon (in blue). BiP protein (yellow) is close the translocon.