Because of its protein-denaturing ability, urea has played a pivotal role in the experimental and conceptual understanding of protein folding and unfolding. The measure of urea's ability to force a protein to unfold is given by the m value, an experimental quantity giving the free energy change for unfolding per molar urea. With the aid of Tanford's transfer model [Tanford C (1964) J Am Chem Soc 86:2050–2059], we use newly obtained group transfer free energies (GTFEs) of protein side-chain and backbone units from water to 1 M urea to account for the m value of urea, and the method reveals the anatomy of protein denaturation in terms of residue-level free energy contributions of groups newly exposed on denaturation. The GTFEs were obtained by accounting for solubility and activity coefficient ratios accompanying the transfer of glycine from water to 1 M urea. Contrary to the opinions of some researchers, the GTFEs show that urea does not denature proteins through favorable interactions with nonpolar side chains; what drives urea-induced protein unfolding is the large favorable interaction of urea with the peptide backbone. Although the m value is said to be proportional to surface area newly exposed on denaturation, only ≈25% of the area favorably contributes to unfolding (because of newly exposed backbone units), with ≈75% modestly opposing urea-induced denaturation (originating from side-chain exposure). Use of the transfer model and newly determined GTFEs achieves the long-sought goal of predicting urea-dependent cooperative protein unfolding energetics at the level of individual amino acid residues.
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机译:由于其具有蛋白质变性能力,尿素在蛋白质折叠和展开的实验和概念理解中发挥了关键作用。尿素迫使蛋白质解折叠的能力的度量由m值给出,m值是一个实验量,给出了每摩尔尿素解折叠的自由能变化的实验量。借助Tanford的转移模型[Tanford C(1964)J Am Chem Soc 86:2050-2059],我们使用了新获得的蛋白质侧链和骨架单元的基团转移自由能(GTFE),从水到1 M尿素再到考虑到尿素的m值,该方法根据变性时新暴露的基团的残留水平自由能贡献揭示了蛋白质变性的解剖结构。通过考虑甘氨酸从水转移到1 M尿素中的溶解度和活度系数比来获得GTFE。与一些研究者的观点相反,GTFEs表明尿素不会通过与非极性侧链的良好相互作用而使蛋白质变性;驱动尿素诱导的蛋白质展开的原因是尿素与肽主链之间的良好相互作用。尽管据说m值与变性时新暴露的表面积成正比,但只有≈25%的面积有利于展开(由于新暴露的骨架单元),而≈75%则适度地反对尿素诱导的变性(起源于侧链曝光)。转移模型和新近确定的GTFE的使用实现了一个长期的目标,即在单个氨基酸残基的水平上预测尿素依赖性合作蛋白的展开能。
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