I have solved a DNA Polymerase crystal structure at 2.15 Ang. with a modified nucleotide. Structure shows 4 molecules in the asymmetric unit. Interestingly, each molecule of the asymmetric unit is different from each other (showing difference in the active site, DNA strand positions, some residue movements with a mechanism for the binding and incorporation of a nucleotide in an active site). For example In molecule 1. Incoming nucleotide is little far from the primer -OH gp and in molecule 4 its perfectly shows an pre-insertion complex, while molecule 2 and 3 are an intermediate stages. The same DNA polymerase with the natural nucleotide was crystallized as one molecule in the asymmetric unit with different crystallization condition (2nd) thus doesn't shows such kind of mechanism. I tried to crystallize the modified incoming nucleotide and DNA polymerase complex in 2nd crystallization condition but it didn't crystallize. Now question is how I can prove that the mechanism is a real mechanism but not a crystal artifact?
This is a very interesting situation you just described. Do you have any kind of idea on the affinity of the natural and modified nucleotides? Perhaps the modified nucleotide allows the observation of intermediate states due to the states being more stable than for the natural nucleotide? Proving this might be tricky though. I suppose NMR spectroscopy could be used to detect chemical perturbations at the relevant amino acid resonances. These might vary as a function of temperature or upon nucleotide titration and thus work out how the polymerase functions. You could also try to measure binding energy/affinity through ITC or SPR. Computational methods might help greatly also.
Have you tried intensive screening for other conditions for your setups? Often if your protein crystallizes in a certain way and you add a substrate that behaves differently, it might not be reproducible with another compound. Maybe it could be possible to detect the intermediates also for the natural nucleotide if crystallized differently. To help this, you could try to generate a panel of mutants with point-mutations in the active site and see if you manage to crystallize those with the natural nucleotide, as personally I think proving the reaction mechanism is to show it using the natural substrate. This has worked for me before with a 2H phosphodiesterase.
These crystals are reproducible, with other same type of modified compounds. We have done some biochemical kinetics with this modified nucleotide. Kinetics shows that Kd is little lower than natural nucleotide while the rate of incorporation of the modified nucleotide is sevearl times slower (don't remember the exact value) than the natural nucleotide. I am trying to crystallize this complex in different condition without any success.
So binding kinetics are slower but the affinity is quite the same? How is your modified nucleotide different to the natural one? Bulkier atoms? Charge difference? Binding kinetics can be monitored through NMR spectroscopy, which might shed some light on which amino acids show altered chemical shifts upon substrate addition. Might be difficult since presumably your protein is quite big as DNA polymerases often are and would anyway require labeling.
I think that targeted mutagenesis might really be helpful in this situation unless you want to test your theory computationally.
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