We all are so very familiar with this picture. The rotating molecule, bound to its immutable aptamer, and the resulting "bonus", which also seems perfectly constant...
I mentioned a few times in this blog that EteRNA simplifies many things. With good reasons, of course. But we will soon need to get past some of these (over)simplifications, so I'd like to give you guys a heads-up.
So, we shall begin with this information acquired from scientific sources (which I need to find back to cite properly here, where the hell did I read that... ah, found it):
The binding energy for FMN to the RNA aptamer is: -8.6 kcal/mol.
Wait, what? Not -4.9?
Let me clarify the previous statement: it means that IF the FMN molecule would stay in place bound to the aptamer permanently, then it would provide -8.6 kcal/mol worth of stabilization to the RNA polymer. And of course, everything is in that "if". Because you guessed it now, that's not (actually, never) the case.
I'd like to invite you to watch this animation I made some time ago:
If the EteRNA graphical interface could be made more realistic, it would represent the situation as a RNA surrounded by floating molecules (instead of bubbles)
It would stay like this for say a bit less than a second, and then, one molecule would dock:
And it would stay attached for maybe a little more than a second, at which point it would undock. Then, the cycle would simply repeat...
Now, if you consider the fact that under specific conditions, the RNA is bound to the FMN in average 57% of the time, then you have no problem figuring out that the average stabilization bonus experienced by the RNA sequence is -8.6 times 57%, which makes (drumroll) about -4.9 kcal/mol. Et voila.
Multiple factors come into play for the amount of time that is spent bound rather than unbound. The static ones are related to the nature of the ligand. The association (Ka) and dissociation (Kd) constants have been determined experimentally.
Also, it is quite obvious that if more molecules are in the vincinity of the binding site, the higher the chance for a successful binding. Which is simply a question of concentration.
In EteRNA, it was decided long ago to use 10mM (millimolar), as a way to help players figure out switches. That concentration corresponds to the familiar -4.9 value we all know (don't forget that it's an average). But this is also a clearly exaggerated dosis, and for practical purposes, it would be good if we could manage to work with less. 200uM (micromolar) is the concentration we should shoot for in our future efforts about switches, and that corresponds to an average bonus of -2.51 kcal/mol.
(*) The 'Identification of a 14mer RNA that recognizes and binds flavin mononucleotide with high affinity' paper mentions that "the original 35mer binds FMN with a KD of ∼500 nM, which corresponds to a ΔGassociation of −36 kJ mol−1". The 35mer mention refers to the PDB entry 1FMN, and the conversion of kJ to kcal (use http://www.cactus2000.de/uk/unit/massear.shtml for instance) gives -8.6