 This video introduces a protein science paper by Mahona Sarkar and Gary Pylac, entitled An Osmalite Mitigates the Destabilizing Effect of Protein Crowding. Hello, I'm Gary Pylac. Osmalites are small organic molecules that stabilize proteins. They are found in all living things. One of their functions is to counter stress, including dehydration-induced stress. The major osmalite in E. coli is glycine beta-ene, which is simply a glycine with three methyl groups on its nitrogen atom, making it a zwitterion at physiological pH. Well, the cellular interior is crowded. The concentration of macromolecules is 300 grams per liter, and they occupy 30% of the volume of the cell. The original view of crowding effects involved only steric repulsions, which implied that crowding can only stabilize proteins. Now, dehydration-induced stress only increases the crowded nature of the cytoplasm. And we know that cells make osmalites in response to dehydration-induced stress. This leads to a puzzle. If crowding is only stabilizing, why would osmalites be required to overcome dehydration-induced stress? Well, now Mahona will discuss our experiments and our conclusions. I'm Mahona. I worked with Gary to understand the simultaneous effects of an osmalite and protein crowding on protein stability for our present paper. We performed NMR-detected amide proton exchange with CI2 as our test protein and different biomolecules as crowders. This technique allows us to observe residue-level stability without any external perturbations such as heat or denature end. We have shown before that CI2 is stabilized by inert synthetic polymers, whereas it's actually destabilized by biologically relevant protein crowders. Here, in our protein science paper, we have tested the stability of CI2 in buffer, under crowding by proteins from E. coli cytoplasm, and then add in an osmalite to the crowded system. This table summarizes our results. We measured global stability of CI2 in buffer, in protein lysates, in buffered osmalite solutions, and protein lysates with added osmalite to it. We first observed that protein lysate destabilizes CI2 by 600 calories per mole. This happens mostly by non-specific interactions with the backbone of our test protein. Adding osmalites shields these non-specific interactions. This results in an increase in CI2 stability by 200 calories per mole. We could now say that crowding is more dominated by steric repulsion's effect. The effects now are similar to those observed in PVP polymer in a previous study. Also in this figure, you can see that the CI2 stability is color-coded by a rainbow, where residues with shades of red are destabilized and blue are stabilized. CI2 changes from red to blue once osmalite is added to the crowded system for almost all residues. We conclude from our work here that osmalites make the test protein CI2 invisible to other proteins present as crowders. Probably now, protein crowders behave as inert crowders and therefore stabilize CI2 by more of a steric repulsion effect. Thank you, Mahouda. I will wrap up by suggesting where the field is going. First, we need to identify the mechanism of what we observed. That is, examine the dependence of the effect on the identity of the osmalite and its concentration. Second, we need to measure the effect of osmalites on proteins in living cells. We have started along these paths by developing a method for measuring stability in living E. coli cells. That paper was published recently in PNAS. Mahouda and Gary's paper is being published in the journal Protein Science. Thank you for watching. Please contact Mahouda or Gary if you have any questions or comments. Happy reading!