title: Structural and thermodynamic characterisation of L94F mutant of horse cytochrome c.
abstract: Mammalian mitochondrial cytochromes c (cyts c) has a conserved Leu94 which is replaced by valine/isoleucine in some lower eukaryotes and prokaryotes. It is expected that nature substituted Leu94 with Val/Ile, for they have similar van der Waals volume and hydrophobicity with a difference in side chain branching only. Reports also suggested the presence of phenylalanine at position 94, which leads to questions: (i) How bulky aromatic amino acid residue fitted at position 94 in cyt c family proteins? (ii) What is the effect of L94F mutation on protein stability and folding? Here, we selected horse cyt-c as a model to answer the second question. We generated L94F mutant of horse cytochrome c and subsequently characterised using far-UV, near-UV and Soret circular dichroism, absorbance, intrinsic and extrinsic ANS (8-anilino-1-napthalenesulfonic acid) fluorescence and dynamic light scattering measurements. We observed that this mutation affects the native state and arrests the protein folding at the molten globule state. Thermal stability of L94F mutant is also measured by spectroscopic techniques and differential scanning calorimetry. The midpoint of thermal denaturation of L94F mutant is 17°C less than wild type. Molecular dynamics simulation study also supports our in vitro observation that this mutant has stable backbone conformation.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
authors.forename: Sabab Hasan
authors.initials: SH
authors.lastname: Khan
authors.affiliation: CSIR-Institute of Genomics and Integrative Biology, South Campus, Sukhdev Vihar, Mathura Road, New Delhi 110025, India.
authors.forename: Atul
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
authors.forename: Amresh
authors.initials: A
authors.lastname: Prakash
authors.affiliation: CSIR-Institute of Genomics and Integrative Biology, South Campus, Sukhdev Vihar, Mathura Road, New Delhi 110025, India.
authors.forename: Bhupesh
authors.initials: B
authors.lastname: Taneja
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
authors.forename: Md Imtiayaz
authors.initials: MI
authors.lastname: Hassan
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India. Electronic address: fahmad@jmi.ac.in.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000302:isolation & purification; D000067493:Dynamic Light Scattering; D006736:Horses; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D050505:Mutant Proteins; D045304:Cytochromes c
keywords: Horse cytochrome c; MD simulation; Molten globule; Pre-molten globule; Protein denaturation; Thermodynamic stability