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Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry
 Kavan, Daniel
CD69 is considered as the marker receptor of activated lymphocytes and is expressed at sites of active immune response. Physiologically it appears in the form of covalently bound homodimer, however after examining its three- dimensional structure we suggested Q93 on one subunit and D88 with E87 on the other one to participate on the inter-subunit interactions. Even more profound intertwining was observed in case of R134 of one subunit with A136 and Y135 on the other one. Therefore Q93, R134 or both were mutated into alanines and showed the monomeric form just in case of double-mutant. This fact influenced significantly also the binding of ligands. While the Kd values for binding of GlcNAc was approximately 10-5 M in case of monomeric form, in case of dimeric form it was 10 times lower and even 100 times lower in case of the longest covalently bound dimers. Although the gel filtration retention time decrease was observed, which could indicate a change in molecular fold, the value of experimentally determined sedimentation coefficient was identical. Moreover neither the comparison of HSQC NMR spectra before and after ligand saturation revealed any significant shifts. Hydrogen deuterium exchange is a chemical process in which a covalently bonded hydrogen atom is replaced by deuterium or vice versa. As...
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