Methanol-stabilized intermediates in the thermal unfolding of ribonuclease A. Characterization by 1H nuclear magnetic resonance.
Methanol-stabilized intermediates in the thermal unfolding of ribonuclease A. Characterization by 1H nuclear magnetic resonance.
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核糖核酸酶 A 热解折叠中的甲醇稳定中间体。通过 1H 核磁共振表征。
DOI：
10.1016/0022-2836(82)90133-4
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发表时间：
1982
影响因子：
5.6
通讯作者：
Fink,AL
中科院分区：
文献类型：
--
作者：
Biringer,RG;Fink,AL
核糖核酸酶A的热去折叠已研究在25，35和50%的甲醇（体积比）的解决方案，使用360 MHz的质子磁共振波谱。一些观察结果表明，在甲醇低温溶剂中的蛋白质的天然结构是非常相似的水溶液中。展开过程的详细分析已经取得了使用的C-2质子的咪唑侧链的四个组氨酸残基。随着变性的进行，新的共振出现，其化学位移既不对应于天然的也不对应于完全未折叠的物种。这些已被分配到特定的希氏残基的选择性氘化研究。热变性转变揭示了在每种溶剂中的多相过程，并且随着甲醇浓度的增加而变得不太合作。变性是完全可逆的，没有滞后的证据。在展开过程中出现的新的共振归因于部分折叠的物种，其通过相对疏水的甲醇的存在而稳定。基于化学位移的温度依赖性和各种共振的相对面积，已经提出了一个详细的事件序列来描述展开过程。关键特征包括两个结构域的初始一般松动，随后上部S-肽区域（残基13至25）远离蛋白质的主体移动，随后部分分离片层结构并完全暴露N-末端螺旋，导致“翼结构域”完全分离，并最终丧失残余片层和螺旋结构。
The thermal unfolding of ribonuclease A has been studied in solutions of 25, 35 and 50% methanol (v v), using 360 MHz proton magnetic resonance spectroscopy. Several observations indicate that the native structure of the protein in methanol cryosolvents is very similar to that in aqueous solution. A detailed analysis of the unfolding process has been made using the C-2 protons of the imidazole side-chains of the four histidine residues. As denaturation proceeds new resonances appear, whose chemical shifts correspond to neither native nor fully unfolded species. These have been assigned to particular His residues by selective deuteration studies. The thermal denaturation transitions reveal a multiphasic process in each of the solvents, and become less co-operative with increasing concentrations of methanol. The denaturation is fully reversible with no evidence of hysteresis. The new resonances that appear during the unfolding process are attributed to partially folded species, which are stabilized by the presence of the relatively hydrophobic methanol. Based on the temperature dependence of the chemical shifts and the relative areas of the various resonances, a detailed sequence of events has been proposed to describe the unfolding process. Key features include the initial general loosening of the two domains, the subsequent movement of the upper S-peptide region (residues 13 to 25) away from the main body of the protein, followed by partial separation of the sheet structure and full exposure of the N-terminal helix, leading to complete separation of the “winged domains”, and ultimately the loss of the residual sheet and helix structure.