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

• Published in: Archives of biochemistry and biophysics 2017-11, Vol.633, p.40-49
• Main Authors: Khan, Sabab Hasan, Islam, Asimul, Hassan, Md. Imtaiyaz, Sharma, Sujata, Singh, Tej Pal, Ahmad, Faizan
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Substitution; Animals; Binding Sites; Cloning, Molecular; Cytochromes c - chemistry; Cytochromes c - genetics; Cytochromes c - metabolism; Enzyme Stability; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Horse cytochrome c; Horses; Isoleucine - chemistry; Isoleucine - metabolism; Kinetics; Leucine - chemistry; Leucine - metabolism; Molten globule; Mutation; Protein Binding; Protein denaturation; Protein Interaction Domains and Motifs; Protein stability; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Site-directed mutagenesis; Substrate Specificity; Thermodynamics; Valine - chemistry; Valine - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2017.08.015

A sequence alignment of horse cytochrome c (cyt c) with all known cyts c shows that Leu at position 94 is conserved, except in 14 species which have either Val or Ile at this position. It is also known that Leu94 of the mammalian cyt c plays an important role in folding and stability. The important question here is as to what will happen in terms of folding and stability if Leu94 of the mammalian cyt c is substituted by Val or Ile. To answer this question, we introduced natural substitutes of Leu94 by Val and Ile in horse cyt c. The purified L94V and L94I mutants under native condition (pH 6.0, 25 °C) were characterized using far-UV, near-UV and Soret- circular dichroism, visible absorbance, Trp and ANS (1-anilino-8-napthaline sulphonate) fluorescence and dynamic light scattering measurements. Furthermore, stability parameters Tm (mid-point of denaturation) and ΔGD0 (Gibbs free energy change at 25 °C) were also determined using spectroscopic and differential scanning calorimetric methods. All these measurements led us to conclude that both mutants exist as molten globule and are less stable than the wild-type protein. These observations are supported well by examining the structure of horse cyt c (PDB ID, 1HRC). •In all mammalian cytochromes c, position 94 is occupied by Leu only.•Mutation of Leu94 by Val and Ile (natural substitutes) leads to misfolding of the horse cytochrome c.•Both mutants (L94V and L94I) have structural characteristics of molten globule at pH 6 and 25 °C.•L94V and L94I mutants are less stable than the wild type protein both in terms of Tm and ΔGD0.