Dao-pin S, Nicholson H, Baase W A, Zhang X J, Wozniak J A, Matthews B W
Department of Physics, University of Oregon, Eugene 97403.
Ciba Found Symp. 1991;161:52-62. doi: 10.1002/9780470514146.ch4.
The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein. Such studies can provide quantitative information on the contributions made by different types of interactions including hydrogen bonds, hydrophobic interactions, salt bridges and disulphide bridges. To determine the contribution of long-range electrostatic interactions a combination of charge-change mutations was used to reduce the overall formal charge on T4 lysozyme at neutral pH from +9 to +1 units. Such changes in charge were found to have little effect on the stability of the molecule. Salt bridges engineered on the surface of the protein also were found to contribute little to stability. In contrast, the introduction of acidic groups designed to interact with the partial positive charges at the N-termini of alpha-helices consistently increased the stability of the protein. It is argued that this difference between electrostatic salt-bridge interactions and electrostatic 'helix-dipole' interactions lies in the entropic cost of bringing together the interacting partners. In an attempt to simplify the folding problem, and also to further investigate the helix propensity of different amino acids, a series of alanines was introduced within an alpha-helix of T4 lysozyme. The resultant protein not only folds normally but is also more stable than the wild-type enzyme, adding further support to recent evidence that alanine is a helix-favouring amino acid.
来自噬菌体T4的溶菌酶正被用作一个模型系统，以确定单个氨基酸在典型球状蛋白质折叠和稳定性中的作用。这类研究能够提供关于不同类型相互作用（包括氢键、疏水相互作用、盐桥和二硫键）所做贡献的定量信息。为了确定长程静电相互作用的贡献，使用了电荷变化突变的组合，以使T4溶菌酶在中性pH下的总形式电荷从+9减少到+1单位。发现这种电荷变化对分子的稳定性影响很小。在蛋白质表面设计的盐桥对稳定性的贡献也很小。相比之下，引入旨在与α-螺旋N端的部分正电荷相互作用的酸性基团，始终会增加蛋白质的稳定性。有人认为，静电盐桥相互作用和静电“螺旋偶极”相互作用之间的这种差异在于将相互作用的伙伴聚集在一起的熵成本。为了简化折叠问题，并进一步研究不同氨基酸的螺旋倾向，在T4溶菌酶的一个α-螺旋内引入了一系列丙氨酸。所得蛋白质不仅能正常折叠，而且比野生型酶更稳定，这进一步支持了最近的证据，即丙氨酸是一种有利于形成螺旋的氨基酸。
