Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A
Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A
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DOI：
10.1021/bi001945w
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发表时间：
2001-01-09
期刊：
影响因子：
2.9
通讯作者：
Gussakovsky, EE
中科院分区：
文献类型：
--
作者：
Navon, A;Ittah, V;Gussakovsky, EE
本研究旨在区分牛胰腺核糖裂解酶A（RNase A）可逆热变性过程中的局部去折叠和整体去折叠。通过9个突变体的稳态和时间分辨荧光监测局部展开，其中每个突变体中的单个色氨酸被取代为野生型残基。通过远紫外圆二色性和紫外吸光度监测全局解折叠。所有突变体（除F8 W和D38 W外）均表现出较高的比酶活，其远紫外圆二色性光谱与野生型RNase A非常接近，表明色氨酸取代不影响任何突变体（除K1 W和Y 92 W外）在20 ℃折叠条件下的结构。与野生型RNase A一样，各种突变体在pH 5下表现出可逆的协同热去折叠转变，转变温度比野生型转变低2.5-11 ℃，如通过远紫外CD或UV吸光度检测到的。即使在80 degreesC下，远高于所有RNase A突变体的合作转变，也保持了相当数量的二级和三级结构。这些研究表明，随着温度的升高，核糖核酸酶A的热去折叠转变的两个阶段的机制。首先，在低于主要协同转变温度的温度下，主要疏水核内的长程相互作用减弱，例如，涉及残基Phe-8（在N-末端螺旋中）和Lys-104和Tyr-115（在C-末端β-发夹基序中）的那些。链反转环（残基91-95）的结构在相同的温度范围内松弛。其次，随后的高温协同解折叠转变与二级结构的损失和主要疏水核的三级接触的额外变化有关，例如，涉及分子另一侧的残基Tyr-73、Tyr-76和Asp-38的那些。蛋白质的C-末端环的疏水相互作用通过高温增强，并且可能是负责在略高于主要合作转变的温度下保存Trp-124的局部结构环境。这些结果为热去折叠转变提供了新的线索，总体上支持了伯吉斯和谢拉加的热去折叠假说，并经马西森和谢拉加修改。
This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degreesC. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 OC lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degreesC, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta -hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.