Cooperative Transition in the Conformation of 24-Mer Tarantula Hemocyanin upon Oxygen Binding

Wolfgang Erker,Ute Beister,Heinz Decker

doi:10.1074/jbc.m414516200

Wolfgang Erker, Ute Beister + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m414516200

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Abstract

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.

Highlights

Hemocyanins are respiratory proteins of numerous arthropods and mollusks occurring freely dissolved in the hemolymph [1,2,3]
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 predicted for the allosteric unit

Summary

EXPERIMENTAL PROCEDURES Materials

Hemocyanin of tarantula E. californicum consists of four hexamers and has a molar mass of 1.721⁄7106 g/mol [23, 24]. Protein concentration was calculated from the optical density; an absorption of 1.1 at 278 nm corresponds to a concentration of 1 mg/ml Eurypelma hemocyanin [10]. To label the protein with the fluorescence probe, Prodan solution was added to hemocyanin in buffer and incubated for 1 h at 4 °C. The binding of Prodan to hemocyanin was investigated by fluorescence titration and analyzed according to Mas and Colman [27]. The binding of the dye is connected with an increase of its fluorescence intensity. A linearized form describing the measured fluorescence intensities in dependence on the protein concentration is, K. and at high protein concentrations, [P]free can be replaced by [P]total, which is justified in our case (30 nM dye and up to 10.5 ␮M protein, 24-mer). Based on the determined parameters K and Q, the concentration of bound ligand could be calculated according to

F F0 Ϫ 1

RESULTS

DISCUSSION