Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli
Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli
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DOI：
10.1002/pro.5560031107
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发表时间：
1994-11
期刊：
影响因子：
8
通讯作者：
G. Georgiou;P. Valax;M. Ostermeier;P. Horowitz
中科院分区：
文献类型：
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作者：
G. Georgiou;P. Valax;M. Ostermeier;P. Horowitz
TEM β-内酰胺酶已被用作理解大肠杆菌中导致包涵体形成的途径的模型。TEM β-内酰胺酶的平衡变性显示，在中间（1.0-1.4 M）浓度的氯化胍（GdmCl）下，中间体失去了酶活性、天然蛋白荧光和UV吸收，但保留了60%的天然圆二色性信号。该物质表现出双-1-苯胺基-8-萘磺酸荧光的大幅增加，表明存在暴露的疏水表面。当TEM β-内酰胺酶在不同初始浓度的GdmCl中解折叠并通过透析重折叠至相同的最终条件时，在1.0-1.5 M范围内的GdmCl初始浓度下观察到活性蛋白产率的明显最小值。结果表明，较低的再活化产率完全是由于非共价连接的聚集体的形成。我们提出TEM β-内酰胺酶的聚集涉及具有部分暴露的疏水表面的紧密状态的缔合。这一假设与我们最近的发现一致，即TEM β-内酰胺酶包涵体含有广泛的二级结构（Przybycien TM，Dunn JP，Valax P，Georgiou G，1994，Protein Eng 7：131-136）。最后，我们还表明，蛋白质聚集增强，在较高的温度下，并在5 mM的二硫苏糖醇的存在下，并通过添加蔗糖抑制。这些条件对体内包涵体的形成产生类似的影响。
The enzyme TEM β‐lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM β‐lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0–1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis‐1‐anilino‐8‐naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM β‐lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0–1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM β‐lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM β‐lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131–136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.