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首页> 外文期刊>Biochemistry >Local and Long-Range Interactions in the Thermal Unfolding Transition of Bovine Pancreatic Ribonuclease A
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Local and Long-Range Interactions in the Thermal Unfolding Transition of Bovine Pancreatic Ribonuclease A

机译：牛胰核糖核酸酶A的热展开转变中的局部和远距离相互作用。

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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 Kl Wand Y92W) under folding conditions at 20 °C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 °C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 °C, 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 p-hairpin motif). The structure of the chain-reversalloop (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.
机译：进行这项研究以区分牛胰腺核糖核酸酶A（RNase A）可逆热变性中的局部和全局展开。通过九个突变体的稳态和时间分辨荧光来监测局部展开，其中每个突变体都用一个色氨酸代替了野生型残基。通过远紫外线圆二色性和紫外线吸收监测整体展开。除F8W和D38W以外的所有突变体均表现出高的比酶活性，其远紫外CD光谱与野生型RNase A的光谱非常接近，表明色氨酸取代不会影响任何突变体的结构（在20°C的折叠条件下不包括Kl Wand Y92W）。像野生型RNase A一样，各种突变体在pH 5时均表现出可逆的协同热解折叠转变，通过远紫外CD或紫外吸收检测，其转变温度比野生型转变低2.5-11°C。即使在80°C时，也远高于所有RNase A突变体的协同转变，仍保持了大量的二级和三级结构。这些研究表明，随着温度升高，RNase A的热解折叠转变的以下两步机制。首先，在低于主要协同转变温度的温度下，主要疏水性核内的远程相互作用减弱，例如，涉及残基Phe-8（在N端螺旋中）以及Lys-104和Tyr-115（在C末端的p型发夹基序中）。反向链的结构（残基91-95）在相同温度范围内松弛。其次，随后的高温协同展开转变与二级结构的丧失和主要疏水核的三级接触的额外变化有关，例如，涉及疏水基团上的残基Tyr-73，Tyr-76和Asp-38的那些。分子的另一面。蛋白质的C末端环的疏水相互作用通过高温增强，并且可能负责在略高于主要协同转变的温度下保留Trp-124的局部结构环境。结果为热展开转变提供了新的思路，通常支持由Matheson和Scheraga修改的Burgess和Scheraga的热展开假设。

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