Laboratory Exercises Immunoassay for Visualization of Protein-Protein Interactions on Ni-Nitrilotriacetate Support

Hundreds of chemical and physical signals constantly bombard the surface of the cells. Some of these do not enter the cell but, instead, bind to receptors at the cell surface and initiate a flow of information that moves to the cell interior. Stimulation of these receptors by hormones, odorants, or light activates a group of coupling proteins called G-proteins (because they bind GTP) that regulate a variety of enzymes and ion channels [1–3]. G-proteins are made up of three polypeptide subunits, , and , the subunit acting as an enzyme that binds and hydrolyzes GTP. The and subunits form a dimer that dissociates only when denatured. When G-proteins are signaling, they in essence function as two separate units as the signal can be communicated either by the subunit or the dimer. In mammals, there are currently known 20 , 7 , and 11 subunits, and on the basis of sequence similarities of their subunits, G-proteins are grouped into four subfamilies, Gs, Gi/0, Gq, and G12 [4]. The G-protein cycle that transmits the signal from receptor to effector starts when GDP is bound to the subunit that associates with dimer to form an inactive heterotrimer, which binds to a receptor. When a signal stimulates the receptor, it is activated and changes its conformation. The activated receptor activates GDP-liganded subunit that decreases the GDP affinity and increases the affinity for GTP. The dissociation of GDP from the subunit is followed by association of GTP. Once GTP is bound, the subunit assumes its active conformation and dissociates from both receptor and dimer. The activated state lasts until GTP is hydrolyzed to GDP and Pi by intrinsic GTPase activity of the subunit. After cleavage of GTP to GDP, the subunit reassociates with dimer, becomes inactive, and binds to the receptor. The free subunit and dimer each activate target effectors [5, 6]. The functions of G-proteins are primarily regulated by association of GTP with the subunit, hydrolysis of GTP to GDP and Pi, and dissociation of GDP. Besides these low molecular mass regulators of G-protein function, G-proteins are activated by other nonreceptor activators such as AlF4 , nonhydrolyzable analogues of GTP [1], some hydrophobic amines, or amphiphilic peptides [7, 8]. In order to demonstrate protein-protein interaction and to avoid the commonly used kinetic approach, we have developed an immunoassay for visualization of heterotrimeric G-protein association/dissociation between the i2 subunit and 1 2-His 6 dimer overexpressed in sf9 cells. This article describes a biochemistry experiment for undergraduate microbiology students who are formally following lessons in biochemistry and have already completed lessons and experimental exercises on general chemistry, physical chemistry, and cellular biology. The exercise may be used in typical biochemistry and molecular biology laboratories within different professional fields (chemist, biologist, pharmacist, biochemist, microbiologist . . . ) where the protein structure, function, and regulation are involved. The exercise is definitely not suitable for freshmen without any basic and experimental knowledge. In addition, it can also be performed with postgraduate students to refresh their knowledge on particular biochemical/molecular biology techniques. As outline of this exercise is given in Fig. 1.