Deletion of the terminal sequences from cytochromes c

Area of Science:

• Biochemistry
• Structural Biology
• Protein Chemistry

Background:

• Human heart cytochrome c is a crucial protein in cellular respiration.
• Understanding cytochrome c structure-function relationships is vital for metabolic research.
• Limited solubility of modified cytochrome c fragments hinders detailed analysis.

Purpose of the Study:

• To investigate the structural and solubility consequences of terminal deletions in human heart cytochrome c.
• To characterize the conformation and heme environment of truncated cytochrome c fragments.
• To elucidate the role of terminal segments in maintaining protein structure and solubility.

Main Methods:

• Chemical cleavage using cyanogen bromide to generate specific peptide fragments.
• X-ray crystallography to analyze the three-dimensional structure of the hemepeptide.
• Spectroscopic techniques (e.g., UV-Vis, CD) for qualitative and quantitative analysis.
• Hydrodynamic measurements to assess protein size and shape in solution.

Main Results:

• Deletion of the amino-terminal 12 residues (residues 1-12) produced an insoluble hemepeptide (13-104)H.
• The (13-104)H hemepeptide exhibited significant conformational changes compared to native cytochrome c.
• Deletion of the carboxyl-terminal residues (residues 81-104) yielded a soluble hemepeptide (1-80)H.
• The (1-80)H hemepeptide showed spectral and hydrodynamic properties suggesting a stable, encapsulated heme within an apolar cluster.

Conclusions:

• The amino-terminal region of human heart cytochrome c is critical for maintaining protein solubility and native conformation.
• The carboxyl-terminal region contributes to the stability of the heme environment in soluble fragments.
• Heme encapsulation in a hydrophobic core, stabilized by axial ligation, is a key feature of the soluble hemepeptide (1-80)H.