Small‐angle X‐ray scattering reveals differences between the quaternary structures of oxygenated and deoxygenated tarantula hemocyanin

Decker, Heinz; Hartmann, Hermann; Sterner, Reinhard; Schwarz, Erika; Pilz, Ingrid

doi:10.1016/0014-5793(96)00887-3

Cited by 27 publications

(31 citation statements)

References 29 publications

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“…The interhexameric distance as obtained by TEM had to be shortened to be in agreement with the SAXS data. Similar deviations of calculated distance distribution functions p(r) based on TEM from those obtained by SAXS have been reported previously for the tarantula hemocyanin (19). As in the case of the 2ϫ6-meric lobster hemocyanin, the half-molecules of the 2ϫ12-meric tarantula hemocyanin had to be shifted together to yield contacts between the half-molecules and to be in reasonable agreement with the SAXS data (Fig.…”

Section: Discussionsupporting

confidence: 77%

“…The observed differences seem to be small. However, even when the oxy and deoxy states of the 24-meric hemocyanin from the tarantula E. californicum were compared, differences of similar magnitude were obtained (19).…”

Section: Discussionmentioning

confidence: 87%

“…This technique has been applied successfully to monitor changes of the quaternary structure for several cooperative proteins such as hemoglobin and a number of allosteric enzymes (10 -18). Different conformations have also been reported for the oxy-and deoxyhemocyanin from the tarantula Eurypelma californicum from SAXS measurements (19).…”

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confidence: 99%

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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

Hartmann

Lohkamp

Hellmann

et al. 2001

Journal of Biological Chemistry

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Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector L-lactate on the oxygenated quaternary structure of the 2؋6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mM L-lactate resulted in different scattering curves compared with those obtained in the absence of L-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of L-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.Hemocyanins are the oxygen transport proteins of most molluscs and arthropods. They are enormous molecular structures, ranging in molecular mass from 4.5 ϫ 10 5 to more than 10 7 Da; some contain more than 100 oxygen binding sites (1-3). Hemocyanins exhibit cooperative oxygen binding and respond to a variety of allosteric effectors (4 -8). As a consequence of their structural complexity, hemocyanins have proved important in extending our understanding of allostery. For example, explanation of the cooperative oxygen binding by arthropod hemocyanin has required extension of the classical MWC 1 model to the "nesting" model (8, 9), which reflects the hierarchical structure of these proteins. The nesting model requires four states (rT, rT, tR, rR) instead of the two states (T, R) of the MWC model. It makes the prediction that even fully oxygenated hemocyanin can exist in two conformational states. Although the model has served well to explain oxygen binding and its dependence on effector (see Ref. 8, for example), it has never been tested directly. That is, no experiments have been performed to detect the two oxy conformations predicted by the nesting model. To be definitive, such experiments must be carried out in solution, under rigorously controlled oxygenation levels and with a sensitivity sufficient to detect even small conformational changes.A suitable method to detect conformational changes of large proteins in solution is small angle x-ray scattering (SAXS). This technique has been applied successfully to monitor changes of the quaternary structure for several cooperative proteins such as hemoglobin and a number of allosteric enzymes (10 -18). Different conformations have also been reported for the oxy-and deoxyhemocyanin from the tarantula Eurypelma californicum fro...

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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 87%

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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

Hartmann

Lohkamp

Hellmann

et al. 2001

Journal of Biological Chemistry

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“…Thus, the observed cooperativity in native Hc can only be explained by changes in the quaternary structures. Different oxygen-dependent conformations have been shown for various Hc (Decker et al, 1996;Miller, 1982, 1995;Salvato and Beltramini, 1990;Markl and Decker, 1992). In addition, dimerisation of the two hexamers of C. aestuarii Hc results in a higher cooperativity and a stronger Bohr effect (Table 1) indicating a functional coupling between the two hexamers.…”

Section: Discussionmentioning

confidence: 96%

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

Dainese

Muro

Beltramini

et al. 1998

European Journal of Biochemistry

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Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca 2ϩ : 24S accounting for 90% of total hemocyanin and 16S accounting for 10%. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component. The 5S component consists of three monomeric fractions named CaeSS1 (10%), CaeSS2 (50%) and CaeSS3 (40%); the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 have been studied as far as their reassociation properties to form hexamers are concerned. We investigated the oxygen-binding properties of the native form (24S), the mixture of the two 16S forms, the pH-stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggregate. The analysis of O 2 -binding data reveals that 24S-Hc can be well described by the modified Monod Wyman and Changeaux-model (nested MWC-model), while the half-molecules (16S) bind oxygen according to the simple MWC-model. The two hexameric 16S within the dodecameric 24S hemocyanin can be regarded as nested allosteric units. They behave as being functionally coupled in the T-states (tT and rT). In the R-states (tR and rR) the two half-molecules seem to be functionally uncoupled since they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.Keywords : hemocyanin; subunit; heterogeneity; oxygen binding ; allostery ; nesting.Hemocyanins (Hc) are giant oligomeric copper-containing proteins. They occur freely dissolved in the hemolymph and are responsible for oxygen transport in many arthropods and molluscs (Ghiretti, 1962;Préaux and Gielens, 1984;Salvato and Beltramini, 1990; van Holde and Miller, 1995). Arthropod Hc occur in the hemolymph as aggregates of the basic 16S unit (24S, 37S, 48S, 53S and 62S). The 16S unit is a hexamer of 5S functional subunits. The specific aggregation forms, present in the hemolymph of the various animals, depend on the species (van Holde and van Bruggen, 1971;Markl, 1986;Ellerton et al., 1983). In the hemolymph of most crustacea, hexamers (16S) or dodecamers (24S) or both are found. However, the biological significance for the occurrence of such different aggregation forms is still controversial (Markl and Decker, 1992). Dissociation in functionally intact subunits can be accomplished by removing Ca 2ϩ and increasing the pH above 9.0 (Ellerton et al., 1983; van Holde and Miller, 1995). Various studies elucidate the subunit composition of crustacean Hc. In general, three subunits are found namely a, b and c subunits (Bak and Beintema, 1987;Jekel et al., 1988; Neuteboom et al., 1992;Markl, 1986). They are differently expressed in the various species (Mangum, 1993) and are necessary to guarantee the specific aggregation state (Markl and Decker, 1992). Hc bind oxygen as peroxide in a µϪη 2 :η 2 -coordination mode between the two Cu(II) ions ; while subunit...

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“…The persistence of two states at high oxygen concentrations is due to a limited allosteric range and experimentally confirmed for arthropod hemocyanins (29). DISCUSSION The hemocyanin of tarantula E. californicum shows highly cooperative oxygen binding, possess 24 oxygen-binding sites, and is able to adopt different conformations (10,30,31). However, transitions between these conformations are scarcely investigated so far.…”

Section: Oxygen Binding Curvesmentioning

confidence: 99%

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

Erker

Beister

Decker

2005

Journal of Biological Chemistry

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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 ϳ2, 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.

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Small‐angle X‐ray scattering reveals differences between the quaternary structures of oxygenated and deoxygenated tarantula hemocyanin

Cited by 27 publications

References 29 publications

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

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

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

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

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