Cooperative transition in the conformation of 24-mer tarantula hemocyanin upon oxygen binding.
Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula hemocyanin. Oxygen binding of the hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding curves recorded with tryptophan and Prodan emission revealed that structural changes in tarantula hemocyanin lag behind oxygen binding at the beginning of oxygenation. Analyses based on the nested two-state model, which describes cooperative oxygen binding of hemocyanins, indicated that the transition monitored by Prodan emission is closely related to one of the four conformations (rR) predicted for the allosteric unit. Earlier, the allosteric unit of tarantula hemocyanin was found to be the 12-mer half-molecule. Here, fluorescence titration revealed that the number of Prodan binding sites/24-mer tarantula hemocyanin is approximately 2, matching the number of allosteric units/hemocyanin. Based on the agreement between oxygen binding curves and fluorescence titration we concluded that Prodan monitors a conformational transition of the allosteric unit.
Erker
Wolfgang
W
Department of Physical Chemistry, Johannes Gutenberg University, Mainz, Welderweg 11, 55099 Mainz, Germany. erker@mail.uni-mainz.de
Beister
Ute
U
Decker
Heinz
H
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D006433:Hemocyanins / Q000737:chemistry*; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008968:Molecular Conformation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010313:Partial Pressure; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013112:Spiders; D014364:Tryptophan / Q000737:chemistry
D046911:Macromolecular Substances; C038517:prodan; D014364:Tryptophan; D006433:Hemocyanins; D015081:2-Naphthylamine; D010100:Oxygen