A study on the role of evolutionarily invariant leucine 32 of cytochrome c.
Abstract: To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation constant by more than 400-fold for the ferrous complex. In contrast, replacement of leucine 35 with isoleucine seems to increase it only by a small degree. Since both leucine 32 and leucine 35 are completely buried within the structure, hydrophobic interaction would not explain this striking difference. However, thermodynamic analyses and absorption spectra of the ferric complex have indicated that replacement with norvaline of leucine 32 increases both delta H and delta S (more positive) associated with formation of an intermediate three-fragment complex and decreases both delta H and delta S (more negative) associated with transformation from the intermediate to the ground state, resulting in weakening the methionine 80--S--heme-Fe bond formed in the latter step. Taking the results together with the fragment exchange studies on the ferrous complex and available evidence, we suggest that the interaction involving leucine 32 would be coupled not only with the methionine 80--S--heme-Fe bond but also with the energy state of other distant residues such as tryptophan 59, generating extra energy for modulating the binding of the complex, i.e. the force of folding. In contrast, leucine 35 would be less important even if it were involved in such coupling.
Publication Date: 1986-02-25 PubMed ID: 3005260
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- Journal Article
Summary
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This paper studies the impact of substituting leucine 32 in horse cytochrome c, a protein involved in cell respiration, with other amino acids. It finds that this substitution significantly affects the protein’s binding abilities and structure, suggesting the significant role of leucine 32 in energy generation and protein folding.
Methodology
- The researchers synthesized analogs of residues 28-38, which contain a replaced amino acid at positions 32 or 35, using Merrifield’s method.
- They examined the ability of these analogs to bind to the two-fragment complex of heme fragment and apofragment through gel filtration and equilibrium dialysis.
Findings
- Substituting leucine 32 with isoleucine increased the dissociation constant by over 400-fold for the ferrous complex, which greatly diminished the binding ability of the protein.
- In contrast, replacing leucine 35 with isoleucine only modestly increased the dissociation constant.
- Since both leucine 32 and 35 are completely covered within the protein structure, the difference in their roles wouldn’t be explained by their hydrophobic interaction.
- Thermodynamic analyses indicated that replacing leucine 32 with another amino acid affected both the delta H and delta S associated with the formation of the intermediate cytochrome c complex and its conversion to the ground state.
- The researchers proposed that the interaction involving leucine 32 is linked not only to the bond formed with methionine 80 but also to the energy state of other far-off residues, creating supplementary energy that influences the binding and folding of the complex.
- In contrast, leucine 35 seems to be less vital to these processes.
Implications
- This research emphasizes the important role of leucine 32 in the structure and energy production in cytochrome c proteins.
- Finding that substituting this specific amino acid leads to significant changes in protein structure and function could contribute to a better understanding of cellular energy production mechanisms and potentially inform drug development targeting these processes.
Cite This Article
APA
Juillerat MA, Taniuchi H.
(1986).
A study on the role of evolutionarily invariant leucine 32 of cytochrome c.
J Biol Chem, 261(6), 2697-2711.
Publication
Researcher Affiliations
MeSH Terms
- Amino Acid Sequence
- Animals
- Chromatography, Gel
- Cytochrome c Group
- Dialysis
- Horses
- Leucine
- Mathematics
- Models, Chemical
- Structure-Activity Relationship
- Thermodynamics
Citations
This article has been cited 3 times.- Lett CM, Guillemette JG. Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions.. Biochem J 2002 Mar 1;362(Pt 2):281-7.
- Taniuchi H, Shi Y, San Miguel GI, Ferretti JA, Mack JW, Fisher A, Shah M, Schechter AN, Shiloach J. A study of the influence of the hydrophobic core residues of yeast iso-2-cytochrome c on phosphate binding: a probe of the hydrophobic core-surface charge interactions.. J Protein Chem 2001 Apr;20(3):203-15.
- Shaw WV. Protein engineering. The design, synthesis and characterization of factitious proteins.. Biochem J 1987 Aug 15;246(1):1-17.
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