Lab Report #22: Unexpected Lab Results

What we found in the lab wasn't what we expected -- but it can tell us how to move forward. And that is science in action.

- Why design symmetric assemblies?
- Crystal structure of a symmetric design by NinjaGreg

- Symmetric design puzzles
- Protein binder design puzzles
- Electron density puzzles

- A symmetric D2 tetramer design by an anonymous player boasts a unique fold, but a lot of hydrophobic surface area that could cause trouble. See for yourself in the July Design of the Month sandbox puzzle.

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(Thu, 07/01/2021 - 13:41  |  6 comments)
Susume's picture
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Rosetta energy of trimer docking vs tetramer?

Does Rosetta report different ddG of docking for the tetramer vs trimer? We can't really test diffferent symmetries for our designs, but if we could, would foldit tell us when we need to discourage off-target dockings?

bkoep's picture
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Rosetta prefers trimer

Good question!

We can calculate the Rosetta DDG of an interface by computing the Rosetta energy when two chains are bound, and then recomputing the energy when they are apart. The difference in these computed energies (the Rosetta DDG of binding) predicts the actual change in free energy upon binding.

In this case it seems that the Rosetta DDG of the trimer interface is more negative than any of the tetramer interfaces. This means that Rosetta predicts the trimer should be more stable than the tetramer from the crystal structure!

Most likely, we can chalk this up to inaccuracies in the Rosetta score function. However, we should be a little cautious of this result, because we may not have the whole story. The crystal structure comes from x-ray diffraction data at a resolution of only 3.2 A. We can be pretty sure about the overall shape of the backbone in our crystal structure, but we can't be confident in the configuration of all the sidechains. And that could have a significant effect on DDG.

jeff101's picture
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NinjaGreg's D2 Tetramer

Congratulations to NinjaGreg for his good design.
It is interesting that despite being designed as a 
C3 trimer, it wound up crystallizing as a D2 tetramer.
This makes me wonder if you could find intermediates
in solution that are dimers or trimers of the protein.

I'm thinking that as monomers collide with each other
in solution, sometimes two will collide in just the 
right way so they stick together and form a dimer.
I think it is much less likely that three or more
monomers will collide in just the right way to stick
together and directly form trimers, tetramers, etc. 
I think it is more likely that first dimers are formed. 
Some of these dimers then collide with monomers to form 
trimers. Later, the trimers collide with monomers to 
form tetramers. There might even be collisions between 
dimers that lead to the formation of tetramers. One 
could even imagine pentamers or hexamers forming and 
then breaking apart. I'd imagine that a variety of 
these complexes could all coexist in solution in 
equilibrium with each other, with some complexes being 
more common than others. I also think that if you vary 
the starting concentration of monomer, you can get 
different populations of all the complexes formed. 
Perhaps at certain concentrations of monomer, the 
trimeric form is more common than the D2 tetramer

What do you think?

bkoep's picture
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Those are great points! You're absolutely right, at room temperature we expect that all of the molecules in solution are constantly interchanging between different assemblies.

If we assume that the solution is at equilibrium, then we can ignore all of the possible assembly pathways and the rates of association/dissociation. At equilibrium we know ultimately that the proportions of the different assemblies is related to their relative free energy (just like proportions of folded/misfolded molecules discussed in this blog post).

It's also true there could be some dependence on the concentration of protein molecules. This relates to one of the downsides of x-ray crystallography experiments: protein crystals typically form only at extremely high concentrations, which can sometimes lead to crystal structures that do not represent the structure of the protein at lower concentrations.

jeff101's picture
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What is the lowest protein concentration that will crystallize?

Now that you know NinjaGreg's protein can crystallize,
you could try crystallizing it from lower protein 
concentrations. I'd imagine that at very low protein 
concentrations, most of the protein would be in the
monomeric form. As you raised the protein concentration,
the amount forming dimer, trimer, tetramer, etc. would
gradually increase. At some point, the protein would
crystallize. If you found the lowest protein concentration
from which crystals could form, perhaps the crystals 
formed would be more pure (like all trimer instead of a 
mix of trimer and tetramer). Don't purer crystals give
higher resolution in x-ray crystallography?

Also, aren't there other techniques to determine the size
of protein complexes in solution? For example, could Gel 
Electrophoresis do it? How about Size-Exclusion 
Chromatography or UV-Vis Spectroscopy? I'm guessing you
know of others.

See below for details:

horowsah's picture
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difficulty in determining how many

One of the things people often find hard to believe about biochemistry is that the problem of determining how many monomers are in a complex is often a remarkably hard problem. It seems simple on the surface, but so much of biochemistry is built on uniqueness. So when you have multiple copies of the same thing, it often gets hard to tell how many there are, no matter how many different techniques you use. All of the techniques have weaknesses. To make it harder, it could be that multiple answers are correct, depending on the conditions used. And on the crystallography question- while some proteins will crystallize differently in different conditions and at different concentrations, it's pretty rare to find it. Getting a protein to crystallize is usually a problem of finding a needle in a haystack, and so to get it to crystallize two different ways is often the equivalent of trying to find two needles in two haystacks, but usually the second haystack is much bigger than the first.

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Developed by: UW Center for Game Science, UW Institute for Protein Design, Northeastern University, Vanderbilt University Meiler Lab, UC Davis
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