Let there be {
if (RhoGTP + Effector == ProteinComplex) {
Luc1 + Luc2 = ActiveLuciferase;
LIGHT = 1; }
else if (RhoGTP + GAP == RhoGDP) {
RhoGDP + Effector = NoProteinComplex;
LIGHT = 0; }
else if (RhoGDP + GEF == RhoGTP) {
RhoGTP + Effector = ProteinComplex;
Luc1 + Luc2 = ActiveLuciferase;
LIGHT = 1; }
else
LIGHT = 0;
cout << LIGHT << endl;
}
LIGHT?
Clay Clark
By Clay Clark – @biochemprof
Small GTPases are important for regulation of a host of cellular functions, from gene transcription and other signaling cascades to cell motility, proliferation, and vesicle transport.
In their simplest form, the proteins work as binary switches, where the “on-state” depends on the type of nucleotide bound. For example, with guanosine triphosphate (GTP) bound, the protein forms a conformation that allows it to interact with other proteins (called effectors) that, in turn, convey a molecular signal. In contrast, when the terminal phosphate is hydrolyzed by the GTPase, such that guanosine diphosphate (GDP) is bound, the protein forms a conformation considered the “off-state.” In this form, the protein does not interact with other signaling proteins.
Here’s a short movie that shows a morph between the two nucleotide-bound states of the small GTPase Arf: Arf Conformational Change
The process starts a so-called “molecular clock” in which the amplitude of the signal depends on the length of time GTP is bound to the GTPase because the GTP determines whether the protein is in the “on-state.”
In order to control the “molecular clock”, the cell has evolved other proteins that function to control the nucleotide-bound state of the GTPase. On the one hand, guanine exchange factors (GEFs) facilitate the release of GDP and the binding of GTP to the small GTPase. This, in effect, results in the conversion to the “on-state.” On the other hand, GTPase activating proteins (GAPs) facilitate the hydrolysis of GTP by the GTPase. Because this hydrolysis converts the GTP to GDP, the effect is to transform the GTPase into the “off-state.”
Now, to complicate matters further, the terms “small GTPase”, ”GEF” and “GAP” are general terms that describe classes of proteins. There can be more than one protein in each class – that is, dozens of small GTPases, dozens of GEFs, and dozens of GAPs. This is further complicated by the fact that each GTPase will interact with some GEFs and GAPS, but not all. In other words, there is specificity in the interactions.
Sorting out which GTPases interact with which GEFs and GAPs in the cell has been problematic. Because a given GTPase will interact with multiple GEFs and GAPs, and with differing affinities, the “molecular-clock” is actually a complex network of protein-protein interactions.
So, how does one study these interactions? Until recently there were two primary assays used. In the first, the binding and/or hydrolysis of GTP is monitored either through radioactivity of the GTP (where the terminal phosphate is radioactive) or through fluorescence emission of a GTP analogue. In the second, pull-down assays are used to capture proteins that bind to the GTP-bound GTPase (that is, the “on-state”). Each method has advantages and disadvantages. Some provide high sensitivity but require handling of radioactivity, while others are more environmentally friendly but suffer from lower sensitivity (see the review listed below by Jaiswal and coworkers). In addition, not all of the assays can be used in context of the cellular milieu. Ideally, an assay would be environmentally friendly, retain high sensitivity, and be useful for protein interaction studies in cells.
Basic Design of the Split Luciferase Assay
Such an assay was recently developed by Anderson and Hamann, from the Biology Department at Bemidji State University, and published in Biochemical Journal. The assay is based on a split-luciferase gene in which two parts of the firefly luciferase gene sequence are fused to the genes of potentially interacting proteins. When the fused proteins are made, the two luciferase parts are not active until the fusion partners interact. In that case, the two luciferase units are brought together to form an active protein. The activity of luciferase, and thus the interaction of the fusion partners, can be measured by the amount of light produced.
Anderson and Hamann tested the system using several Rho GTPases and their effector proteins. The Rho family of GTPases is important because Rho GTPases regulate the assembly of actin structures in the cell. In this publication, three RhoGTPases were fused with the second luciferase fragment (called Luc2), and the GTPase binding regions of several effectors were fused to the first luciferase fragment (called Luc1) (see the Figure above). This experimental design allowed the authors to test each of the three Rho GTPases with several effectors and thus to examine binding specificity.
The sensitivity of the assay was shown to be comparable to that of the radioactivity-based assay, as it is effective at low nano molar concentrations of protein. In addition, RhoGTPase-Effector combinations that are known to occur were recapitulated in these experiments. The assay also was sensitive to the presence the GAP and GEF factors, that is, the nucleotide-bound state of the RhoGTPase. Finally, the assay was tested with mammalian cell lysates after expression of exogenous GEFs. The authors found that the GEFs in the lysates were capable of stimulating GDP/GTP exchange and that the assay could be used to distinguish mutated GEFs that are inactive versus wild-type GEF (active).
The assay is an exciting new addition to the arsenal of tools to study small GTPases. Although the assay was developed with Rho GTPases, it could, in principle, be used with a wide array of small GTPases. It could be used to examine novel GEF and GAP specificities by examining a panel of proteins. It could be used to screen small molecule drug libraries to identify compounds that affect GTPase activity. It could be used to examine protein networks in the cell following extracellular stimulation, exogenous expression of GTPase effectors, or functional consequences of specific mutations.
Sensitivity, specificity, environmentally friendly, and adaptable.
References:
Anderson, E., & Hamann, M. (2012). Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system. Biochemical Journal, 441 (3), 869-879 DOI: 10.1042/BJ20111111
Jaiswal M, Dubey BN, Koessmeier KT, Gremer L, & Ahmadian MR (2012). Biochemical assays to characterize Rho GTPases. Methods in molecular biology (Clifton, N.J.), 827, 37-58 PMID: 22144266
Massoud, T., Paulmurugan, R., De, A., Ray, P., & Gambhir, S. (2007). Reporter gene imaging of protein–protein interactions in living subjects Current Opinion in Biotechnology, 18 (1), 31-37 DOI: 10.1016/j.copbio.2007.01.007
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