Dear Anastasis,
I am sorry for a very late reply.
Thank you very much for the detailed description :)
Regards
Kavya
> Ok, lets start from the basics. ;-)
>
> Y= x/(kD+x)
>
> … maximal binding=1 for simplicity
> Y is the fraction bound
> x is the ligand cocnentration
> and kD is your KD
>
> so …
>
> x= (Y * KD) / (1 - Y)
>
> … you can plot that and stick it in your desk really, for a few K_D
>
> … and to make a long story short, the numbers are more or less exactly as
> Sebastian says ...
>
> However, life is more complicated.
>
> When a protein interacts with a ligand and the reaction has reached
> equilibrium (which in most cases happens in the milliseconds to several
> seconds scale) , although we call that a “steady state” this is till a
> dynamic interaction. Molecules of the protein and the ligand associate and
> disassociate all the time, but the system is in equilibrium.
>
> P+L <—> PL
>
> This equilibrium can then be described by the association rate, or K_on,
> that depends on the absolute concentration of the reactants and is
> expressed as events per molarity per time M^(-1)sec^(-1)
>
> The disassociation or K_off, does not depend on the concentration of the
> reactants, but is only a function of time, e.g. how many complexes
> disassociate per second, and is expressed in sec^(-1)
>
> The infamous disassociation constant we have all been talking of, K_D, is
> then the K_off/K_on, thus has Molarity as a unit, and it tells you the
> concentration in which as many complexes are made as they associate as a
> function of time. In practice, when you measure association with one
> reactant in very low concentration and titrate the other one one, K_D is
> the point that you have 50% of “saturation”.
>
> Now, back to your question: when you crystallise you change the system I
> just described, so Kon, Koff, and thus KD will change. So, you cannot just
> predict. However, some simple rules:
>
> 1. Better have more ligand than protein, as the protein needs to be
> “saturated", and you will never do that with less ligand than protein ...
> 1. You better be at least 3x or preferably 10x over the K_D with your
> ligand, to make sure most of your mixture is bound.
> 2. You better have a low K_off. If a K_off is a minute you have a good
> chance to capture that in a crystal, if its 10ms tough luck: transient
> complexes are hard to crystallise.
>
> Lazy Sunday afternoon - sorry for the long essay and sorry for some
> oversimplifications I made and some seasoned biochemists might be
> chuckling about ;-)
>
> A.
>
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