Effect of conservative mutations (L94V and L94I) on the structure and stability of horse cytochrome c.

This research investigates the impact of specific mutations in the structure of horse cytochrome c, where leucine at position 94 is replaced by either valine or isoleucine. The results suggest that these mutations cause the protein to become less stable than the original form.

Introduction and Background

• The researchers noticed that the leucine (Leu) at position 94 in horse cytochrome c (cyt c) protein is highly conserved – it’s almost always there, except in 14 species that have a valine (Val) or isoleucine (Ile) at that position.
• Previous studies indicated that this Leu94 plays a significant role in the folding and stability of the cyt c protein.
• The main question posed in the study was how replacing Leu94 with Val or Ile in horse cyt c would affect the protein’s folding and stability.

Methodology

• The researchers made the exact mutations they wanted to study – replacing Leu94 with Val (L94V mutation) and Ile (L94I mutation) in horse cyt c protein.
• They then studied these mutant proteins using a variety of methods including far-UV, near-UV and Soret circular dichroism, visible absorbance, fluorescence of Trp and ANS (1-anilino-8-napthaline sulphonate), as well as dynamic light scattering measurements.
• Furthermore, they analysed the stability of the mutant proteins by determining their mid-point of denaturation (T) and Gibbs free energy change (ΔG) at 25°C.

Findings

• The analyses indicated that both L94V and L94I mutations exist as “molten globule” – a term indicating a state in which the protein is not fully structured and somewhat unstable.
• These mutants were also found to be less stable than the wild-type (original) protein.
• The researchers found that the observations were consistent with the structure of horse cyt c model (PDB ID, 1HRC).