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Lecture 28

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 November 14 2012 Lecture 28 Dr. Shock There is quite a large conformational change that a GTPase protein undergoes when it becomes GTP bound (vs. GDP bound) • The presence of the negative phosphate group greatly tightens up the structure through hydrogen bonding allowing it to react different than in its GDP bound state, where it’s relatively loose and unreactive • Phosphorylation does not always change the conformation of a protein, sometimes, the negative charge of the phosphate is only meant as a tag to be recognized by other factors How would you analyze the GTPases? For example, how would you make a mutation similar to the ones for the kinases to see how the pathways work either without the pathway or with the pathway always on. You could mutate 1 amino acid which would prevent hydrolysis of the GTP, leading to the pathway being active all the time (constitutive) Similarly, you could make another mutation which would prevent release of the GDP (i.e. making the protein preferentially bind GDP over GTP) • This would turn the pathway off permanently • Interestingly, this mutation is dominant negative, meaning it’s stronger than a loss of function mutation • Just one copy of the mutant gene is able to produce the loss of function, whereas typical loss of function mutations require a -/- genotype to see the phenotype It’s dominant negative because the GDP bound form of the GTP binds GEFs; because the GEFs can no longer replace the GDP with a GTP, it will remain permanently bound to the mutant GTPase • Remember, there are a lot of GTPase but relatively few GEFs because they’re reusable, you will quickly use up all the GEFS by binding with very high affinity to the mutant GDP bound GTPase • This is a very powerful genetic technique because you don’t even need to produce a double negative mutant to observe the desired 1 th BIOL 300 November 14 2012 Lecture 28 Dr. Shock phenotype We saw before the ligand-receptor binding curve follows a first-order reaction curve (the green curve) • However, what the cell want is a very quick switch- like reaction (like the curve in black) despite the very gradual increases in ligand concentration • Therefore, there must be some kind of amplification in the cell for the ligand-receptor curve to produce a much steeper physiological response curve There are various ways of amplifying a signal through allosteric cooperativity in second messenger proteins with two or more subunits • Allosteric cooperativity means that binding of one substrate to a second messenger protein facilitates binding of the other substrates • For example, in this 4 subunit protein, binding of the 1 st substrate to the active site is the hardest step and represents the least steep part of the response curve • However, once the 1 ligand is bound, it changes the conformation of the whole protein slightly to facilitate binding of the second substrate rd rd • Similarly, the sethnd facilitates the 3 and the 3 facilitates the 4 , which produces a fully active second messenger • Thus for every substrate bound, the curve will become steeper This is a very common topic in biology and is seen everywhere. Another method for amplification is a positive feedback loop: • For example, we have an active enzyme here which activates a second messenger (like ATP  cAMP) • In the event of a positive feedback loop, this second messenger would go back and activate the enzyme that made it to produce more and more second messenger The signal also has to be terminated once the transduction is complete • The receptor gets temporarily removed from the plasma membrane by being internalized into a vesicle which is brought to an endosome, where the receptor-ligand association can no longer occur • Alternatively, you can also degrade the receptor by internalizing it into a lysosome (through formation of an endosome) 2 th BIOL 300 November 14 2012 Lecture 28 Dr. Shock • A choice is made at the endosome stage whether to recycle the contents to the plasma membrane or to progress to a lysosome to degrade the contents • The receptor can also be inactivated by various means, for example, by binding a protein • You could also inactivate any of the downstream mechanisms to terminate the signal even though the ligand is still bound • Finally, you could also produce an inhibitory protein to shut down the pathway So far, we have isolated both the ligand and the receptor we are interested in, and we have found a phenotype in a model organism which occurs when the pathway is shut down We now need to identify the components which make up this pathway. One way to do this is through forward genetics, which involves ra
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