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Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications
L M Sampaleanu1, A R Davidson, C Graham
1Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Protein Science : a Publication of the Protein Society
|March 26, 1999
Summary
Duck delta1 crystallin lost enzyme activity due to domain 1 changes, while delta2 retained it. Hybrid proteins revealed domain 1 is key for restoring argininosuccinate lyase (ASL) activity, suggesting structural adaptation in delta crystallins.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Delta-crystallins are major lens proteins in birds and reptiles, homologous to argininosuccinate lyase (ASL).
- Duck lens has delta1 and delta2 crystallins; delta2 is functional ASL, while delta1 is inactive despite high sequence identity.
- This study investigates the structural basis for delta1's enzymatic inactivity and the functional divergence between delta1 and delta2.
Purpose of the Study:
- To determine the role of individual structural domains in the enzymatic activity of duck delta-crystallins.
- To understand the molecular mechanisms underlying the loss of argininosuccinate lyase (ASL) activity in delta1 crystallin.
- To compare the thermodynamic stability of duck delta-crystallins and human ASL.
Main Methods:
- Construction and analysis of five hybrid proteins (chimeras) between duck delta1 and delta2 crystallins.
- Kinetic analysis to assess the catalytic activity of the hybrid proteins.
- Thermodynamic characterization using unfolding measurements (deltaGs) for delta1, delta2, and human ASL (hASL).
Main Results:
- Kinetic analysis demonstrated that only domain 1 is essential for restoring ASL activity to inactive delta1 crystallin.
- Amino acid substitutions in domain 2 may influence substrate binding.
- Thermodynamic data revealed delta crystallins are less stable than hASL, with delta1 being the least stable (deltaGs: delta1=57.25, delta2=63.13, hASL=70.71 kcal mol(-1)).
Conclusions:
- Domain 1 is solely responsible for the loss of catalytic activity in duck delta1 crystallin.
- The reduced stability of delta crystallins might be an adaptation for their structural role in the eye lens, facilitating protein-protein and protein-solvent interactions.
- Gene recruitment of ASL to a structural protein involved altered functional and stability properties.