Related Experiment Video
Updated: Aug 9, 2026
10:50
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Interactions between yeast iso-1-cytochrome c and its peroxidase
1Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA. gary_pielak@unc.edu
Biochemistry
|January 10, 2001
Summary
This study investigated yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP) interactions. Findings reveal that specific protein interfaces and nonpolar interactions are crucial for stable complex formation and stability.
Area of Science:
- Biochemistry
- Structural Biology
- Protein-protein interactions
Background:
- Yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP) form essential complexes.
- Understanding these interactions is key to deciphering electron transfer mechanisms.
Purpose of the Study:
- To investigate the thermodynamic and structural basis of Cc-CcP complex formation.
- To probe the role of specific residues and nonpolar interactions at the protein interface.
Main Methods:
- Isothermal titration calorimetry (ITC) was employed to study 19 different Cc-CcP complexes.
- Site-directed mutagenesis was used to create CcP and Cc variants.
- Double-mutant cycles were utilized to assess residue coupling energies.
Main Results:
- High-affinity binding sites for Cc and CcP were confirmed to be at the crystallographically defined interface.
- Substitution of charged residues with alanine increased binding enthalpy but affected stability based on location.
- Deletion of methyl groups impacted binding enthalpy and free energy, highlighting the role of hydrophobic interactions.
- Analysis of residue pairs indicated that many substitutions induce complex rearrangements.
Conclusions:
- The study confirms the importance of the defined interface and nonpolar interactions in stabilizing Cc-CcP complexes.
- Protein interface mutations can lead to significant structural rearrangements within the complex.
- Thermodynamic and structural data provide insights into the molecular mechanisms governing Cc-CcP interactions.
Related Concept Videos
01:28Yeast Signaling
Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
01:15Pyruvate Oxidation
After glycolysis, the charged pyruvate molecules enter the mitochondria via active transport and undergo three enzymatic reactions. These reactions ensure that pyruvate can enter the next metabolic pathway so that energy stored in the pyruvate molecules can be harnessed by the cells.
First, the enzyme pyruvate dehydrogenase removes the carboxyl group from pyruvate and releases it as carbon dioxide. The stripped molecule is then oxidized and releases electrons, which are then picked up by NAD+...
First, the enzyme pyruvate dehydrogenase removes the carboxyl group from pyruvate and releases it as carbon dioxide. The stripped molecule is then oxidized and releases electrons, which are then picked up by NAD+...
01:30The Electron Transport Chain
The electron transport chain or oxidative phosphorylation is an exothermic process in which free energy released during electron transfer reactions is coupled to ATP synthesis. This process is a significant source of energy in aerobic cells, and therefore inhibitors of the electron transport chain can be detrimental to the cell's metabolic processes.
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q in...
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q in...
01:24Peroxisomes
Peroxisomes are specialized organelles present in fungi, plant, and animal cells. It can vary in number, size, morphology, and activity depending on the type of tissue and the nutritional state of the cell. For example, cells with active lipid metabolism, such as adipocytes, neurons, and hepatocytes, have more peroxisomes than other cells in the body. Besides their primary role in breaking down complex organic molecules, peroxisomes can also synthesize specific macromolecules and participate in...
01:17Oxidation of Phenols to Quinones
In the presence of oxidizing agents, phenols are oxidized to quinones. Quinones can be easily reduced back to phenols using mild reducing agents. The electron-donating hydroxyl group enhances the reactivity of the aromatic ring, enabling oxidation of the ring even in the absence of an α hydrogen.
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox property is crucial in...
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox property is crucial in...
01:43Electron Transport Chain: Complex III and IV
During the electron transport chain, electrons from NADH and FADH2 are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping...