Khan Sabab Hasan, Kumar Atul, Prakash Amresh, Taneja Bhupesh, Islam Asimul, Hassan Md Imtiayaz, Ahmad Faizan
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
CSIR-Institute of Genomics and Integrative Biology, South Campus, Sukhdev Vihar, Mathura Road, New Delhi 110025, India.
Int J Biol Macromol. 2016 Nov;92:202-212. doi: 10.1016/j.ijbiomac.2016.06.096. Epub 2016 Jul 1.
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.
哺乳动物线粒体细胞色素c(细胞色素c)有一个保守的亮氨酸94,在一些低等真核生物和原核生物中被缬氨酸/异亮氨酸取代。预计自然界用缬氨酸/异亮氨酸取代亮氨酸94,因为它们具有相似的范德华体积和疏水性,只是侧链分支不同。报告还表明在94位存在苯丙氨酸,这引发了一些问题:(i)在细胞色素c家族蛋白的94位,体积较大的芳香族氨基酸残基是如何适配的?(ii)亮氨酸94突变为苯丙氨酸对蛋白质稳定性和折叠有什么影响?在这里,我们选择马细胞色素c作为模型来回答第二个问题。我们构建了马细胞色素c的亮氨酸94突变为苯丙氨酸的突变体,随后使用远紫外、近紫外和索雷特圆二色性、吸光度、内在和外在的1-苯胺基-8-萘磺酸(ANS)荧光以及动态光散射测量对其进行表征。我们观察到这种突变影响天然状态,并使蛋白质折叠停滞在熔球状态。还通过光谱技术和差示扫描量热法测量了亮氨酸94突变为苯丙氨酸的突变体的热稳定性。亮氨酸94突变为苯丙氨酸的突变体的热变性中点比野生型低17°C。分子动力学模拟研究也支持我们的体外观察结果,即该突变体具有稳定的主链构象。