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Lecture

Lecture 27

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Department
Biology (Sci)
Course
BIOL 300
Professor
Siegfried Hekimi
Semester
Fall

Description
BIOL 300 November 12 2012h Lecture 27 Dr. Shock Now, we have isolated the ligand and the receptor, but we want to know how well they are able to bind to one another, i.e. the affinity. A ligand-receptor association is normally a non-covalent interaction (through H bonds, ionic, etc.). Nonetheless, it can be treated as an equilibrium reaction with the receptor and ligand as the reactants, and the receptor-ligand complex as the product • K onuld be a measurement of how well the receptor-ligand complex is formed, while K offwould be a measurement of their level of dissociation • Therefore, K aon K offare inverses of one another • When you have more ligand, the result will be more signaling, which can be seen in the equation above by Le Chatelier’s principle • You can also increase the amount of receptor to increase the amount of signaling to have the same response (more signaling) • The cell often upregulates the amount of receptors to increase signaling • At one point, this reaction will come to an equilibrium where the forward and backward rates are equal; it is only at equilibrium that we can calculate a value for the affinity of the receptor for the ligand • This value is known as K , tde dissociation factor, which is the given by the concentration of free receptors, multiplies by the concentration of free ligands, divided by the concentration of the receptor-ligand complex • Note the work “free” means not complexed • We need to take this at equilibrium so it is independent of the total concentrations of the receptor and ligand • K das a unit of molarity (used to measure concentrations), and gives the affinity of the ligand- receptor complex • A higher K wduld mean you have a lot of free reactants and not a lot of the receptor-ligand complex, showing a low affinity • Therefore, a lower K wodld mean more of the complex (i.e. a higher affinity) • When talking about K values for receptor-ligand interactions, we usually deal with a millimolar d (mM) to a nanomolar (nM) range • Note the special case when K is dqual to the concentration of free ligand; this means exactly half of the receptors are bound to ligand • This essentially tells you at which concentrations the ligands become effective, because most signaling pathways are only turned on when half of the receptors are ligand-bound How do we measure the dissociation constant experimentally upon isolation of a receptor and its ligand? • You don’t necessarily have all of the concentrations needed to calculate it You usually start out with the concentration of the ligand, which you purified; in this example, we’ll talk about insulin, which binds to an insulin receptor • You can vary this concentration to your liking You can then add this insulin to cells; you know the number of cells in your colony by counting them. You will then wait a few minutes to reach equilibrium, and then wash off the excess insulin, and measure the amount of bound insulin by a scintillator. 1 th BIOL 300 November 12 2012 Lecture 27 Dr. Shock You then have to subtract from this total bound insulin the amount of non-specific binding, which increases linearly as you increase concentrations of ligand in order to get the specific binding of the insulin to the insulin receptors • In order to minimize nonspecific binding, we usually test insulin on cell lines that do not naturally express the insulin receptor From this, you are able to draw a binding curve: • On the x axis are concentrations of radioactively labeled insulin, which you vary • Labelling allows you to see measure insulin levels using a special machine • On the y axis would be the amount of bound insulin measured by a scintillator (i.e. the total binding curve), as well as the amount of nonspecific binding, which as we said increases linearly, and then the different of the two, which is the specific binding curve (what we’re interested in) • You should see a curve which starts off steep and then eventually plateaus at high concentrations (a typical 1 order chemical reaction) • K dan thenste inferred because of a few assumptions: • 1 , we assume that at the point where the curve plateaus, all the receptors are bound • 2 , since we know exactly how much insulin you put in, you can calculate the number of receptors on each cell (or in the entire colony) • The K can be taken by looking at the Y value corresponding to 50% of receptors occupied, and d look for the corresponding insulin concentration • This is because, as we said before, when K equads the concentration of ligand, half of the receptors are occupied • You could theoretically calculate K frod any point on the graph, but then you would have to take into account the other concentration values; therefore, 50% receptors bound is the easiest to calculate After calculating the specific binding curve, you can then, using a gene reporter construct, calculate a physiological response curve (i.e. through levels of gene transcription of a reporter gene like beta- galactosidase which turns blue and is easy to see) • The physiological response curve is much steeper than the receptor-ligand binding curve; at about 50% bound receptor concentration, the response is about 80% of its maximum value • Therefore, the signaling pathway is turned on when about half of the ligand is receptor-bound • K dherefore gives the approximate 2 th BIOL 300 November 12 2012 Lecture 27 Dr. Shock concentration at which the ligand is active This is useful even in everyday life, for example in pregnancy tests • Scientists do a lot of research to make a test with the right sensitivity (i.e. low amount of false positives) by isolating the necessary compounds and the K d • Pregnancy tests involve measurements of growth hormones If you have a very low K (high affinity) you can be almo
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