Hydrogen exchange in thermally denatured ribonuclease A.
Hydrogen exchange in thermally denatured ribonuclease A.
复制标题
热变性核糖核酸酶 A 中的氢交换。
DOI：
10.1021/bi00105a014
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Baldwin,RL
中科院分区：
文献类型：
--
作者：
Robertson,AD;Baldwin,RL
斯坦福大学医学院生物化学系，斯坦福，加利福尼亚州 94305 收稿日期：1991 年 5 月 1 日；修订稿于 1991 年 8 月 6 日收到摘要：氢交换已用于测试热变性核糖核酸酶 A (RNase A) 中是否存在非随机结构。使用骤冷流方法和 2D* H NMR 光谱来测量 36 个骨架酰胺质子 (NH) 在 65°C 和 pH*（在 D20 中测量的未校正 pH）值范围为 1.5 至 3.8 时的交换率。结果表明，交换与无序多肽的预测大致相同[Molday, RS, Englander, S. W., & Kallen, R. G.(1972) Biochemistry 11, 150-158]；因此，我们无法检测到热变性 RNase A 的任何稳定氢键结构。然而，两项观察结果表明，应谨慎看待预测的速率。首先，我们发现 Molday 等人（1972）提出的近似值之一，即缬氨酸 NH 的交换与丙氨酸 NH 的交换相似，必须进行修改；缬氨酸 NH 的交换速率大约慢 4 倍。其次，交换的最低 pH 值往往会比预测值低 0.45 个 pH 单位。这些结果与 Roder 及其同事关于牛胰腺胰蛋白酶抑制剂的结果一致 [参见 Roder, H.、Wagner, G. 和 Wüthrich, K.(1985) Biochemistry 24, 7407-7411 中的表 I]，预测和观察到的 pH 最小值之间不一致的根源尚不清楚，但可能是在低 pH 下这些蛋白质上的高净正电荷。与一些其他热解折叠蛋白质一样，热变性核糖核酸酶 A 在远紫外区域显示出显着的圆二色性光谱 [Labhardt, AM (1982) J. Mol.生物。 157、331-355]。通过添加盐酸胍，该光谱可降低至较低水平。造成该光谱的残余结构的性质尚不清楚。我们的结果表明，天然核糖核酸酶 A 的三螺旋形成的螺旋不是稳定的，这可以对酰胺质子交换提供可测量的保护。^) 现代蛋白质化学的主要目标之一是了解蛋白质热力学稳定性的结构基础。对于大多数蛋白质来说，在平衡状态下只有两种状态，即天然状态和变性状态，并且这些状态之间的适度自由能差异决定了蛋白质的稳定性（Becktel＆Schelman，1987）。 X 射线晶体学和最近的 NMR1 光谱提供了有关天然蛋白质结构的高分辨率信息，该信息具有 f 这项工作得到了美国国立卫生研究院 (GM) 的支持
Biochemistry Department, Stanford University School of Medicine, Stanford, California 94305 Received May 1, 1991; Revised Manuscript Received August 6, 1991 abstract: Hydrogen exchange has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A). Quenched-flow methods and 2D* H NMR spectroscopy were used to measure exchange rates for 36 backboneamide protons (NHs) at 65 C and at pH*(uncorrected pH measured inD20) values rangingfrom 1.5 to 3.8. The results show that exchange is approximately that predicted for a disordered polypeptide [Molday, RS, Englander, S. W., & Kallen, R. G.(1972) Biochemistry 11, 150-158]; we thus are unable to detect any stable hydrogen-bonded structure inthermally denatured RNase A. Two observations suggest, however, that the predicted rates should be viewed with some caution. First, we discovered that one of the approximations made by Molday et al.(1972), that exchange for valine NHs is similar to that for alanine NHs, had to be modified; theexchange rates for valine NHs are about 4-fold slower. Second, the pH minima for exchange tend to fall at lowerpH values than predicted, by as much as 0.45 pH units. These results are in accord with those of Roder and co-workers for bovine pancreatic trypsin inhibitor [see Table I in Roder, H., Wagner, G., & Wüthrich, K.(1985) Biochemistry 24, 7407-7411], The origin of the disagreement between predicted and observed pH minima is unknown but may be the high net positive charge on these proteins at low pH. In common with some other thermally unfolded proteins, heat-denatured ribonuclease A shows a significant circular dichroism spectrum in the far-ultraviolet region [Labhardt, AM (1982) J. Mol. Biol. 157, 331-355]. This spectrum is reduced to a low level by adding guanidine hydrochloride. The nature of the residual structure responsible for this spectrum is not known. Our results show that it is not stable helix formation by the three-helices of nativeribonuclease A, which would give measurable protection against amide proton exchange.^) ne of the primary goals of modern protein chemistry is to understand the structural basis for the thermodynamic stability of proteins. For most proteins, only two states, the native and denatured states, are populated to a significant extent at equilibrium and it is the modest free energy difference between these states that determines the stability of a protein (Becktel & Schellman, 1987). X-ray crystallography and, more recently, NMR1 spectroscopy provide high-resolution information about thestructure of native proteins, which has f This work was supported by the National Institutes of Health (GM
等度反相高效液相色谱法测定大鼠肝脏中的腺嘌呤核苷酸。
DOI：
10.1016/0160-5402(85)90032-4
发表时间：
1985
期刊：
Journal of pharmacological methods
影响因子：
--
作者：
Dills,RL;Klaassen,CD
通讯作者：
Klaassen,CD
DOI：
--
发表时间：
1986
期刊：
Journal of Pharmacological Methods
影响因子：
--
作者：
S. Pradhan;R. Copeland
通讯作者：
R. Copeland