Mutations improving the folding of phage P22 tailspike protein affect its receptor binding activity
Mutations improving the folding of phage P22 tailspike protein affect its receptor binding activity
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DOI：
10.1006/jmbi.1999.3165
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发表时间：
1999-10-29
影响因子：
5.6
通讯作者：
Seckler, R
中科院分区：
文献类型：
--
作者：
Baxa, U;Steinbacher, S;Seckler, R
沙门氏菌噬菌体P22尾刺蛋白中的四个先前分离的突变被用于研究蛋白质稳定性、折叠和功能之间的关系。尾刺蛋白结合并水解沙门氏菌脂多糖中的重复O-抗原结构。已知四个突变（V331 G、V331 A、A334 V、A334 I）增加折叠效率，并且其中两个（在位置331处）也增加蛋白质的热稳定性。八糖包含两个重复单元的O-抗原从两个不同的沙门氏菌菌株被用来分析受体结合功能的突变蛋白。它们的内鼠李糖苷酶的酶活性测定与援助的荧光标记的十二糖。发现V331 A和V331 G都强烈影响O-抗原结合。突变蛋白的八糖结合亲和力降低了10倍和200倍，分别对应于标准结合自由能的17%和36%的损失。334位的两个突变仅轻微影响O-抗原结合（Δ Δ G（B）（0）接近1 kJ/mol），但这些突变降低了蛋白的热稳定性。观察到的对内切糖苷酶活性的影响完全解释了底物结合的变化，这表明两种突变都不影响催化速率。所有四种突变体的晶体结构被确定为2.0埃的分辨率。除了部分或完全缺失的侧链，与野生型蛋白质结构相比，在位置331处的突变体没有发现显著的变化，而A334 V和A334 I中突变位点周围的小但显著的骨架置换可以解释观察到的热不稳定。(C)北京：科学出版社.
Four previously isolated mutations in Salmonella phage P22 tailspike protein were used to study the relationship between protein stability, folding, and function. Tailspike protein binds and hydrolyzes the repetitive O-antigen structure in Salmonella Lipopolysaccharide. Four mutations (V331G, V331A, A334V, A334I) are known to increase the folding efficiency, and two of them (at position 331) also increase the thermal stability of the protein. Octasaccharides comprising two repeating units of the O-antigens from two different Salmonella strains were employed to analyze the receptor binding function of the mutant proteins. Their endorhamnosidase enzymatic activity was assayed with the aid of a fluorescence-labeled dodecasaccharide. Both V331A and V331G were found to strongly affect O-antigen binding. Octasaccharide binding affinities of the mutant proteins are reduced tenfold and 200-fold, corresponding to a loss of 17% and 36% of the standard free energy of binding, respectively Both mutations at position 334 affected O-antigen binding only slightly (Delta Delta G(B)(0) approximate to 1 kJ/mol), but these mutations reduce the thermal stability of the protein. The observed effects on the endoglycosidase activity are fully explained by the changes in substrate binding, suggesting that neither of the mutations affect the catalytic rate. Crystal structures of all four mutants were determined to a resolution of 2.0 Angstrom. Except for the partly or completely missing side-chain, no significant changes compared to the wild-type protein structure were found for the mutants at position 331, whereas a small but significant backbone displacement around the mutation site in A334V and A334I may explain the observed thermal destabilization. (C) 1999 Academic Press.