Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix.

This study uses 2D nuclear magnetic resonance techniques to investigate and assign the protons in sequential amino acids of the N-terminal helix in horse ferrocytochrome c, a protein. The findings suggest a regular alpha helix structure between specific amino acids and a disorder at the protein’s N-terminus.

Research Methodology

• The researchers used the two-dimensional nuclear magnetic resonance (2D NMR) to study the 1H resonances of the first 11 sequential amino acids within the N-terminal helix of horse ferrocytochrome c.
• All the main-chain protons from lysine-5 through to alanine-15 and many side-chain protons were assigned. This was done through various spectroscopy techniques.

Spectroscopy Techniques

• J-Correlated Spectroscopy (COSY) was used to differentiate protons on neighboring bonds and identify amino acid types. This is a technique which allows scientists to look at the interaction between atomic nuclei in a molecule.
• Nuclear Overhauser Effect Spectroscopy (NOESY) was used to define spatially contiguous protons and determine which amino acids were sequence neighbors. This nuclear magnetic resonance technique provides information about the distances between atoms in a molecule.
• Relayed Coherence Spectroscopy (Relay COSY) helped resolve any uncertainties in intraresidue J-coupled connectivities and interresidue NOE connectivities. This method contributed to the study without needing specific knowledge of the solution structure.

Findings and Implications

• The pattern of Nuclear Overhauser Effects (NOEs) suggested the presence of a regular alpha helix structure between glycine-6 and lysine-13. This alpha-helix is a common secondary structure seen in proteins.
• Hydrogen bonding seemed to continue through to alanine-15, indicating a continuation of the alpha helical structure.
• The researchers observed disorder occurring at the N-terminus. The N-terminus refers to the start of a protein structure in terms of its amino acid sequence.
• There were no indications of significant spin diffusion among the backbone amide and alpha-protons of this protein, even at the longest NOE mixing time used, which was 140 milliseconds.

Conclusion

• The research has provided insights into the structure of horse ferrocytochrome c, highlighting a regular alpha-helix structure between specific amino acids and disorder at the N-terminus. These findings could be critical for understanding the function of this protein.