Active-site disruption in native Limulus hemocyanin and its subunits by disulfide-bond reductants: a chemical probe for the study of structure-function relationships in the hemocyanins
| Autor: | Richard W. Topham, Celia Bonaventura, Amy Westcott, Gregory Cole, Shirley Tesh, Danielle Mercatante |
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| Rok vydání: | 1998 |
| Předmět: |
Models
Molecular Protein Folding Protein Conformation Protein subunit medicine.medical_treatment Biophysics In Vitro Techniques Biochemistry Dithiothreitol chemistry.chemical_compound Structure-Activity Relationship Protein structure Horseshoe Crabs medicine Molecule Animals Disulfides Protein disulfide-isomerase Molecular Biology Binding Sites biology Chemistry Sulfhydryl Reagents Active site Hemocyanin Oxygen Crystallography Kinetics Hemocyanins biology.protein Calcium Oxygen binding Copper |
| Zdroj: | Archives of biochemistry and biophysics. 352(1) |
| ISSN: | 0003-9861 |
| Popis: | The crystal structure analysis of Subunit II of Limulus hemocyanin has shown that its polypeptide chain is folded into three distinct structural domains. The oxygen-binding, dinuclear copper center is located deep in the core of Domain 2. Two disulfide bonds are located in a bridging domain, Domain 3. These disulfide bonds are remote from the oxygen-binding site, but are positioned so that they could affect its stability. When the disulfide bonds are broken by dithiothreitol or other disulfide-bond reductants, the 340-nm absorption band, associated with oxygen binding, is lost. Disulfide-bond reductants also cause the loss of the oxygen-binding capacity of all seven of the other subunits of Limulus hemocyanin. Thus, disulfide bonding is a general feature of the Limulus hemocyanin subunits that is important to the maintenance of the physiologically effective geometry of the oxygen-binding site. The rate of loss of oxygen-binding capacity, however, is highly dependent on subunit type, aggregation state, and protein conformation. Evidence that protein conformation markedly affects the rate of disruption of the oxygen-binding site comes from the finding that the addition of dithiothreitol to fully oxygenated samples results in a slow initial loss of oxygen-binding capacity followed by an appreciably faster reaction rate. In contrast, in the deoxygenated conformation, the reaction rate is monophasic and never attains the faster rates observed for oxygenated samples. When the disulfide bonds are broken and oxygen-binding capacity is lost, there is subunit-specific variability in the extent of polypeptide-chain unfolding, subunit aggregation, and loss of active-site copper ions. When the disulfide-bond reductant is removed by dialysis so that disulfide bonds can re-form, there is also subunit-specific variability in the extent of restoration of oxygen-binding capacity. Complete restoration of structure and function as the disulfide bonds re-form occurs only for the 48-subunit native molecule, whose architecture is stabilized by bound Ca 2+ and extensive intersubunit contacts. We have found a similar loss of oxygen-binding capacity upon breaking disulfide bonds in a number of other arthropod and mollusc hemocyanins, suggesting that the active site of Limulus hemocyanin is not unique in its dependence upon intact disulfides. The results presented in this paper suggest that disulfide-bond reduction may provide a simple, but powerful, chemical tool with which to probe internal and environmental factors that govern physiologically important structure–function relationships in the hemocyanins. |
| Databáze: | OpenAIRE |
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