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Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c
Insights
Researchers created functional fragments of horse heart cytochrome c by cleaving the protein and reassembling heme and apofragments. These fragments, with high binding affinity, reveal structural insights comparable to Pseudomonas aeruginosa cytochrome c551.
Area of Science:
- Biochemistry
- Protein Chemistry
- Structural Biology
Background:
- Horse heart cytochrome c is a crucial protein in electron transport.
- Understanding protein fragment interactions is key to protein engineering and function.
- Previous studies have explored cytochrome c structure-function relationships.
Purpose of the Study:
- To prepare and characterize noncovalent complexes of cytochrome c fragments.
- To identify specific apofragments and heme fragments that can reassemble.
- To investigate the biological activity and binding affinity of reconstituted cytochrome c complexes.
Main Methods:
- Preparation of a noncovalent complex from horse heart cytochrome c apoprotein and a heme fragment.
- Limited trypsin digestion to remove redundant portions of the ferrous complex.
- Isolation and identification of four apofragments and one heme fragment.
- Demonstration of ordered ferric complex formation using tryptophan fluorescence quenching and a cytochrome b2 assay.
- Estimation of apparent dissociation constants.
Main Results:
- Successfully prepared and characterized four distinct apofragments ((39-104), (40-104), (54-104), (56-104)) and one heme fragment ((1-53)H).
- Demonstrated the formation of ordered ferric complexes with high binding affinity (Kd < 3 x 10(-7) M).
- Reconstituted complexes regained biological activity in a cytochrome b2 assay.
- Identified a cleavage region (residues 38-57) crucial for maintaining structural integrity.
Conclusions:
- The study successfully generated functional cytochrome c fragments, demonstrating the feasibility of protein fragment reassembly.
- The identified cleavage region (residues 38-57) is critical for maintaining the ordered structure of cytochrome c.
- These findings provide insights into protein structural flexibility and evolutionary modifications, correlating with deletions in Pseudomonas aeruginosa cytochrome c551.
Abstract:
A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).