Analysis Of Steadystate Proteinprotein Interaction By Means Of

Many studies are designed to identify steady-state interactions between proteins. RET-techniques, if performed appropriately, offer great sensitivity toward identification of whether RET between two tagged proteins can be detected. However, if the aim of a study is to test for specificity of the observed RET, as well as quantify these interactions, the process is far more complicated. In the following paragraph, the major problems will be discussed in greater detail in order to help to set guidelines for future studies.

How does one determine whether a RET signal between two proteins is specific? It is critically important to use appropriate controls for determining the amount of unspecific RET. However, in many cases, including those of protein interactions with GPCRs, appropriate controls are difficult to establish: We propose the following three criteria be met for a RET-acceptor-control for it to qualify for the term "appropriate control" : (1) Comparable expression levels of the RET-acceptor-control and the RET acceptor of interest need to be achieved. (2) For membrane proteins, the attachment points of the RET-acceptor-fluorophore on control proteins and proteins of interest should be comparable in terms of parameters such as flexibility of the linker, as well as distance to the membrane surface. (3) The RET-acceptor-control should localize to the identical microdomains of the membrane but should not interact with the protein of interest. If one carefully considers these points, it will be extremely difficult to find controls that qualify as appropriate. For example, with our studies on receptor/G protein interactions, none of the acceptor controls that were used satisfactorily qualified for appropriate when the three criteria are applied.

The second problem is the quantification of protein-protein interactions by RET. RET is proportional to the extent of interaction, consequently for i n vitro studies with purified RET acceptor—and in RET-donor-proteins, the quantification of interaction by means of RET is straightforward: specific RET signals in the absence of RET-acceptor molecules (RETmin—should be 0) as well as in the presence of excess acceptor molecules (RETmax) will be measured. The extent of interaction between donor and acceptor molecule is linearly dependent on the RET signal, with (RETmax—RETmin)/2 corresponding to the half-maximal interaction. When using intact cells, in many cases, this issue is even more difficult. The availability of an appropriate acceptor control is needed to determine the amount of unspecific RET (corresponding to RETmin). Next, RETmax needs to be determined in living cells. This requires titration of the expression levels of acceptor molecules relative to those of the donor molecules. Moderate expression levels of the donor need to be achieved, whereas acceptor molecules should be expressed in great excess over donor molecules. Both high expression of acceptors and low expression of donors is not always easy to achieve. In addition, correct localization of donor and acceptor molecules needs to be controlled. The above-mentioned pitfalls are sometimes difficult to circumvent. Additional methods to determine proteinprotein interactions are still needed in order to ascertain the specificity and extent of steady-state interactions.

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