Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme
Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme
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DOI：
10.1073/pnas.97.1.139
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发表时间：
2000-01-04
影响因子：
11.1
通讯作者：
Bustamante, C
中科院分区：
文献类型：
--
作者：
Yang, GL;Cecconi, C;Bustamante, C
单分子操作方法的最新进展为研究蛋白质折叠问题提供了一种新的方法。这些研究通常是在自然组织为球状结构域线性阵列的分子上进行的。为了将这些技术扩展到研究通常作为单体存在的蛋白质，我们开发了一种在固态下合成蛋白质分子聚合物的方法。通过在噬菌体T4溶菌酶分子在晶体中彼此接触的位置引入半胱氨酸，并利用晶格提供的对齐，我们获得了具有限定极性的聚合物，其长度可达25个分子，并保留酶活性。这些聚合物，然后通过使用修改后的扫描力显微镜机械操作，以表征力诱导的可逆的个别溶菌酶分子的展开。这种方法应该是通用的，并适用于许多其他蛋白质与已知的晶体结构。对于T4溶菌酶，在所使用的牵拉速度下展开单体所需的力为64 +/-16pN。在松弛的1秒内发生重折叠，效率接近100%。力与延伸曲线的分析表明，机械展开过渡遵循两态模型。在1 M盐酸胍中测定的解折叠力表明，在这些条件下，解折叠的活化势垒降低了2 kcal/mol。
Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.