Our lab has a new paper coming out this week in the Journal of Molecular Biology (JMB):
I'm going to talk about a few highlights here, but if you actually want the full article, say so in the comments or email me directly and I'll send you a reprint, because unless you or your university has a subscription to the Journal of Molecular Biology, you'll only be able to see the abstract.
The paper comes primarily from the Ph.D. dissertation of Daniel Deredge and osmotic stress data from John Baker and Kausiki Datta. Daniel got his Ph.D. recently and is now a postdoc at Case Western, John was an undergraduate researcher and is now in Med School at Tulane, Kausiki was a former student who is now a postdoc at University of Oregon.
The paper is quite biothermodynamic, but one of the most exciting elements is this:
For about 30 years, biochemists have known that salt disrupts the binding of proteins to DNA. This makes sense because DNA is highly charged. Proteins bind to DNA more weakly in the presence of salt, and adding salt to a protein-DNA complex can break it apart (dissociate it). Over the past 30 years this has been seen for thousands and thousands of different DNA-binding-proteins. But the opposite effect, where salt actually enhances a protein-DNA interaction, has only been seen once. Our paper constitutes the second time it has ever been seen. The first time was such an anomaly that I and others use it in teaching to illustrate how "universal" (we thought) the effect of salt on protein-DNA binding really is: 10,000 cases of salt inhibiting protein-DNA binding - one case of salt enhancing protein-DNA binding. But now, it's 10,000:2. And here's the weird thing: we predicted, based on our analysis, that all other protein-DNA interactions will also show this reversed effect - if you look for it in the right way.
So: how did we look for it? We used glutamate salts. Most biochemistry is done in NaCl or KCl. Potassium and sodium are the most prevalent physiological cations, so that makes sense. But chloride isn't the major intracellular anion, glutamate is (and yes, that is the glutamate in MSG: monosodium glutamate - we used MPG: monopotassium glutamate, because potassium is the major intra-cellular cation while sodium is the major extracellular cation).
So: If we look at binding as we increase the salt concentration, in those 10,000 previous studies, you always see binding getting weaker as salt increases. We usually look at it with something called a "linkage plot" which is shown below for our two DNA polymerases Klenow and Klentaq:
In this Figure, salt concentration is increasing across the X-axis, so the affinity of the protein for the DNA first goes down (the plots have a negative slope at the beginning), but as Kglutamate is increased to really high concentrations (above 1 molar), the trend reverses and the binding starts getting tighter (the plots shift to an upward slope).
Another way to look at it is called a "salt-addition titration" - shown below:
In this Figure we start with a fully formed protein-DNA complex and add more and more salt to it. In both KCl and Kgluatamate, the first thing that happens is the protein-complex falls apart (the steep negative slope at the beginning of both plots). In KCl, once the protein comes off the DNA, it stays off the DNA (that plot flattens out), but in Kglutamate, eventually the increasing salt causes the protein to rebind to the DNA (the plot rises back up showing that the protein-DNA complex is reforming). This has never been directly observed before for any protein-DNA complex (although it has been theoretically predicted as being possible). We spend a lot of the paper attempting to explain these results with mathematical models (which is what the lines through the data represent).
And so what? Who cares that we made a salt-protein-DNA system go backwards for only the second time in history, and that we packed the paper with equations explaining why it does? Well, here's why we care: 1) It shows that our understanding of protein-DNA binding was incomplete: it really can do something that we (we = all biochemists) thought it could not, so we might want to understand that; and 2) Under the highly crowded conditions in the cell the effective salt concentrations are predicted to actually reach these high levels, so it might be that salt works exactly backwards on protein-DNA interactions in the cell relative to the way we've been thinking it acts for the past few decades. As always, only time and more experiments will tell.
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This binding by DNA polymerase is not sequence-specific, right? Would you think that the glutamate effect might also happen with sequence-specific binding?
How common is glutamate? Is it the major anion in plants, fungi, protists, and bacteria?
@Rosie: Yes, polymerases are not sequence specific, but we think this effect will happen with sequence specific binders too because it is due to the osmolyte properties of glutamate helping enhance the reaction by enhancing the removal of surface water that occurs when the protein and DNA associate. "The Glutamate Effect" has been observed for a couple of decades by a number of different labs: it is characterized by a large increase in binding affinity in glutamate, and has been seen for sequence specific and non-specific proteins -- in this paper we're mostly adding this reversal of salt linkage effect as a previously unrecognized feature of the "The Glutamate Effect", along with attempting to explain it.
@Larry: Its been tested for E. coli and a few other bacteria, and for several eukaryotic cell cultures and so now is generally assumed to be the major intracellular anion across the spectrum, but as you note, that doesn't necessarily mean its true across the spectrum. For the effect we see to happen, the "major intracellular anion" has to have some strong "osmotic" character (has to contribute strongly to changing the water activity) and other anions like aspartate or even fluoride seem to do that, just not chloride (all anions will have some osmotic character, but some will be much stronger).
yes nice."The Glutamate Effect" has been observed for a couple of decades by a number of different labs: it is characterized by a large increase in binding affinity in glutamate, and has been seen for sequence specific and non-specific proteins -- in this paper we're mostly adding this reversal of salt linkage effect as a previously unrecognized feature of the "The Glutamate Effect", along with attempting to explain it.
Since glutamate is a component of proteins, won't its intracellular concentration be regulated differently that other anions like Cl- or OAc-? Do you know what the ratio of glutamate to other anions in cells is and if that ratio changes at different stages of cell growth and division?
@Friendly Scientist: Absolutely. It seems to me that there is a lot more study of and understanding of cation regulation than anion regulation, so it seems like a wide open area, and as you say, it is related to protein synthesis and degradation, also neurotransmission, also intracellular osmotic regulation. I know that it is apparently the "major" free intracellular anion in both E. coli and in mammalian cells, but I don't offhand know its ratio to other anions, or if that has been studied relative to development.