Kumar Sandeep, Sharma Deepak, Kumar Rajesh
School of Chemistry and Biochemistry, Thapar University, Patiala 147004, India.
Council of Scientific and Industrial Research - Institute of Microbial Technology, Sector 39A, Chandigarh, India.
Biochim Biophys Acta. 2014 Mar;1844(3):641-55. doi: 10.1016/j.bbapap.2014.01.012. Epub 2014 Jan 28.
The effect of denaturants on the structural fluctuation of M80-containing Ω-loop of ferrocytochrome c was determined by measuring the rate coefficient of CO-association with ferrocytochrome c under varying concentrations of urea and alkylureas (methylurea (MU), N,N'-dimethylurea (DMU), ethylurea (EU), tetramethylurea (TMU)) at pH7.0, 25°C. As denaturant concentration is increased within the subdenaturing limit, the CO-association reaction is decelerated indicating that subdenaturing concentrations of denaturant reduce the structural fluctuation of the Ω-loop. Structural fluctuation of the Ω-loop is reduced more for urea and least for TMU. Intermolecular docking between horse cytochrome c and denaturant molecule (urea, MU, DMU, EU and TMU) reveals that polyfunctional interactions between the denaturant and different groups of Ω-loop and other part of protein decrease with an increase of alkyl group on urea molecule, which suggests that the decrease in the extent of restricted dynamics of Ω-loop with a corresponding increase of alkyl groups on urea molecule is due to the decrease of denaturant-mediated cross-linking interactions. These denaturant-mediated interactions are expected to reduce the conformational entropy of protein. Analysis of rate-temperature data shows a progressive decrease in conformational entropy of protein in the native to subdenaturing region. Thermodynamic analysis of denaturant (urea, MU, DMU, EU, TMU) effects on the thermal unfolding of ferrocytochrome c reveals that (i) thermodynamic stability of protein decreases with increasing concentration of denaturant or hydrophobicity of urea derivatives, (ii) water activity plays an important role in stabilization of ferrocytochrome c, and (iii) destabilization of ferrocytochrome c by denaturant occurs through the disturbance of hydrophobic interactions and hydrogen-bonding.
在pH7.0、25°C条件下，通过测量不同浓度尿素和烷基脲（甲基脲（MU）、N，N'-二甲基脲（DMU）、乙基脲（EU）、四甲基脲（TMU））存在时，一氧化碳与亚铁细胞色素c结合的速率系数，来确定变性剂对含M80的亚铁细胞色素c的Ω环结构波动的影响。在亚变性极限范围内增加变性剂浓度时，一氧化碳结合反应减速，这表明亚变性浓度的变性剂会降低Ω环的结构波动。对于尿素，Ω环的结构波动降低得更多，而对于TMU则降低得最少。马细胞色素c与变性剂分子（尿素、MU、DMU、EU和TMU）之间的分子对接表明，随着尿素分子上烷基的增加，变性剂与Ω环的不同基团以及蛋白质其他部分之间的多功能相互作用会减少，这表明随着尿素分子上烷基相应增加，Ω环受限动力学程度的降低是由于变性剂介导的交联相互作用减少所致。这些变性剂介导的相互作用预计会降低蛋白质的构象熵。速率-温度数据分析表明，在天然状态到亚变性区域，蛋白质的构象熵逐渐降低。对变性剂（尿素、MU、DMU、EU、TMU）对亚铁细胞色素c热解折叠影响的热力学分析表明：（i）蛋白质的热力学稳定性随变性剂浓度增加或尿素衍生物疏水性增加而降低；（ii）水活性在亚铁细胞色素c的稳定中起重要作用；（iii）变性剂使亚铁细胞色素c不稳定是通过干扰疏水相互作用和氢键实现的。
