Abstract
Many biological processes on the molecular level are associated with membrane-bound receptors or other integral membrane proteins. We have been working with proteins that are domains of receptors, or inhibit their function. The first topic are the T-cell surface glycoprotein receptor CD2 and its counter receptor CD58. The other topic is about antagonists of the integrin adhesion receptor glycoprotein IIbIIIa (GPIIbIIIa) which is found on platelet surfaces. Human CD2 is a glycoprotein, and the carbohydrate of its adhesion domain is crucial for adhesion function. The platelet receptor GPIIbIIIa, a Ca2+ dependent heterodimeric glycoprotein from the integrin family, binds fibrinogen and mediates the aggregation of platelets to form a blood clot. Natural protein antagonists of this receptor have primarily been found in the venum of various snakes, which have been termed disintegrins, and in the saliva of blood-sucking leeches. They contain an Arg-Gly-Asp (RGD) sequence in their active site. Due to the potent antiplatelet effect of these RGD proteins, the structures of their active sites have been of considerable interest for the design of antithromotic drugs.
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Discussion
Ad Bax - First, I would like to complement you on this very fantastic work that you presented. I also have a question about somewhat of a paradox about the glucose of the sugar unit on your protein where you observe obviously very strong increased mobility because of the difference in relaxation rates. Nevertheless, you see this loop folding back. Could this be in a small fraction of the time, the thing folds back and causes the NOE or what is going on?
Gerhard Wagner - That’s a good question, eternal question about the NMR structures. The structure we recorded was of proteins that have an rmsd of 0.3 Å. On the other hand, weknow on the interior of the protein, the aromatic rings rotate. Every rotation of an aromatic ring requires an opening of the structure by at least 1.5 Å. So it’s hard to question how much of the protein is in the well-defined state. In the interior of the protein, it might just be 99% and 1% may be a more open structure. I think it is just a gradual decrease of the folding structure and it may well be possible that 80% of the carbohydrate is folded and that the 20% is in a more open state but it is very difficult to identify by experiment but you just have to be aware of it. You should also know that at room temperature proteins have a small fraction of completely unfolded state but that is known from energy exchange experiments. You just have to be aware of this but it is difficult to find out the fraction of the unfolded part of the carbohydrate.
Bax - On a very qualitative level couldn’t you argue that if the T2 is 5 or 10 times longer then it could be only a very small fraction of the time. Let’s say 20% of the thing would be folded up.
Wagner - The problem is to find out if it is 25% or 60%. It is a good point but it’s difficult to answer.
Bax - In your case it is almost an order of magnitude, isn’t it?
Wagner - That’s possible since these carbohydrates have much fewer proteins to relax with.
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Wagner, G. et al. (1996). NMR Studies of Proteins Involved in Cell Adhesion Processes. In: Rao, B.D.N., Kemple, M.D. (eds) NMR as a Structural Tool for Macromolecules. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0387-9_4
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