Intermediate States of Assembly in the Dissociation of Gastropod Hemocyanin by Hydrostatic Pressure

Abstract

Under physiological conditions, the oxygen-transport protein from the gastropod Megalobulimulus ovatus, an extracellular hemocyanin, is composed of 20 identical subunits organized into a cylindrical structure (M(r) 9 x 10(6); 100 S). It dissociates in the pressure range of 0.4-2.5 kbar, as observed by spectroscopic methods (light scattering and intrinsic fluorescence) and gel filtration. In contrast to what is seen with smaller proteins, especially dimers, the pressure-dissociation curves for hemocyanin show little dependence on concentration, suggesting that native hemocyanin exists as a population of molecules with different free energies of association. The pressure-induced dissociation results from an equilibrium in which each aggregate responds to pressure independently of the others and, at any given pressure, is in one of two states, whole or dissociated, which persists for long times when compared with the duration of the experiment. The subunit-subunit affinity of dissociated hemocyanin is much lower than that of associated subunits, suggesting that a conformational drift of monomers occurs. When hemocyanin undergoes dissociation in the absence of calcium and at high pH (> 7.2), a large fraction of the dissociated products changes to a conformation that generates stable intermediate states of assembly, lacking the ability to fully reassemble into decamers and didecamers. These intermediates consist primarily of dimers (M(r) 900,000), and they bind oxygen reversibly with a higher affinity than the native hemocyanin. The binding of calcium or protons changes the conformation back to the "associable" state, which finally generates the assembled structure. The dissociation process is highly reversible at low pH (6.8-6.0) or in the presence of millimolar concentrations of calcium. At pH 5.7, dissociation is negligible at pressures up to 2.5 kbar. A decrease in pH from 7.6 to 6.6 increases the half-dissociation pressure (p 1/2) by 1.3 kbar, corresponding to a stabilization of 1.35 kcal per mole of subunit. The effects of Ca2+ and H+ may mean that, in vivo, special ionic conditions or other factors are required to be present at the assembly sites of oligomeric proteins such as hemocyanin.

Hemocyanin of the Molluscan Concholepas concholepas Exhibits an Unusual Heterodecameric Array of Subunits

“…In the last paragraph of the second column on Page 2758, we incorrectly referenced a previous work in the following sentence: "These results are consistent with a previous study in hippocampal neurons in which synaptic NMDARs were particularly sensitive to caCREB but not dnCREB (12)." In this previous work (Ref.…”

“…removal of divalent cations (11)(12)(13)(14), pH changes, and the addition of denaturing agents (15,16)), have helped to elucidate its subunit composition and structure. In turn, the uses of immunochemical methods combined with partial proteolytic digestion of the subunits, supported by sequence studies, have allowed researchers to establish structural relationships among different FUs and their location in the polypeptide chain (17)(18)(19)(20)(21)(22).…”

Pressure‐induced molten globule state of cholinesterase

“…These findings can be explained by the protective effect of osmolytes against the denaturing action of pressure. This stabilizing effect which is now well documented [14,[24][25][26] may be interpreted in terms of preferential exclusion of osmolyte molecules from the enzyme surface [27]. The presence of the osmolyte shell strips water molecules of the hydration layer of the enzyme, which in turn counteracts the denaturing effect of pressure.…”

High Hydrostatic Pressure Can Probe the Effects of Functionally Related Ligands on the Quaternary Structures of the Chaperonins GroEL and GroES

“…Although other divalent cations such as Ca 2ϩ are known to stabilize macromolecular assemblies (41,42), the role of Mg 2ϩ as a functional ligand is unique for the GroEL-GroES system (14 -18). The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer.…”