Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole

Abstract

Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole, the maximum wavelength A, , , of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for A, , , of the fully oxygenated haemocyanin were 531.5 nm (pH < 7.8) and 530.0 nm (pH > 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH < 7.2) and 535.2 nm (pH > 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S.Only four amino acids of the four-hexamer were labellcd with 7-chloro-4-nitrobenzo-2-oxa-I ,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d.The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.Haemocyanins are the copper-containing respiratory proteins of the two largest invertebrate phyla, the Arthroooda and the Mollusca [l -31. They are oligomeric proteins of very high molecular mass, attaining 3.4 MDa in the horseshoe crab, Limulus polyphemus, and 9 MDa in the vineyard snail, Helix pomatia. Although amino acid sequence studies have revealed that one of the two copper-binding sites (copper B) has a common origin in molluscs and arthropods, the rest of the sequence, including the other copper-binding site (copper A) is clearly not identical [4]. There is also a profound difference in quaternary structure : molluscan haemocyanins form large cylindrical aggregates of about 30 nm in diameter and contain up to 160 oxygen-binding sites, while arthropodan haemocyanins occur as hexamers or multiples thereof.The arthropodan haemocyanins lend themselves particularly well to the study of allosteric interactions between subunits. All of these proteins contain integral multiples of a basic substructure containing six subunits. The smallest naturally occurring haemocyanins are singly hexameric and the largest consist of eight hexamers. These provide a series of aggregates graded by size which offer many possibilities for comparison. For example in the spider Cupiennius saki, single hexamers and two-hexamers occur concurrently, and in this case, the two-hexamers are significantly more cooperative than the single hexamers [5]. Cooperativity is sometimes very high, so that any effects on subunit interaction can be detected with ease.Here we will be concerned with the four-hexameric haemocyanin of the tarantula, Eurypelma californicum. This haemocyani...

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

“…Other methods also confirmed the applicability of the nested MWC model for arthropod hemocyanins. For the 2ϫ12-meric tarantula hemocyanin two different conformations in the oxy state were found using a fluorescence tag 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-chloride (34). This has also been confirmed by O 2 /CO replacement experiments (35).…”

The Structure of a Functional Unit from the Wall of a Gastropod Hemocyanin Offers a Possible Mechanism for Cooperativity,

“…It can be supposed that the binding of dioxygen will induce conformational changes reducing the accessibility to the active site, i.e., that the tunnel is closed after the dioxygen binding. Spectroscopic evidence for conformational changes accompanying oxygenation of arthropod Hcs has already been described ( , ). The comparison of the two active sites and a superposition of the six histidine residues show that they are homologues.…”

Inversion of the Bohr effect upon oxygen binding to 24-meric tarantula hemocyanin.

“…They all assume the existence of more than two different conformations of the subunits (22)(23)(24)(25)(26)(27). However, up to now the postulated conformational transitions have been detected only indirectly-for example, by following changes in absorbance of bound dyes or changes in fluorescence of covalently attached labels (28)(29)(30)(31)(32). Our results demonstrate that hemocyanin undergoes conformational transitions under physiological conditions-that is, in the absence of nonphysiological buffers or covalent labels.…”