DESIGN AND STRUCTURAL-ANALYSIS OF ALTERNATIVE HYDROPHOBIC CORE PACKING ARRANGEMENTS IN BACTERIOPHAGE-T4 LYSOZYME
DESIGN AND STRUCTURAL-ANALYSIS OF ALTERNATIVE HYDROPHOBIC CORE PACKING ARRANGEMENTS IN BACTERIOPHAGE-T4 LYSOZYME
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DOI：
10.1016/0022-2836(92)90475-y
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发表时间：
1992-04-20
影响因子：
5.6
通讯作者：
MATTHEWS, BW
中科院分区：
文献类型：
--
作者：
HURLEY, JH;BAASE, WA;MATTHEWS, BW
尝试设计了噬菌体T4溶菌酶的改进的核心包装安排。替代的掩埋残基Leu 99，Met 102，Val 111和Phe 153选择使用包装计算和能量最小化。为了测试设计程序，构建了一系列多突变体，最终得到替换L99 F/M102 L/V111 I/F153 L。这些变体使T4溶菌酶的稳定性降低约0至2kcal/mol。确定了许多变体的晶体结构。在Val 111被Ile取代的变体中，α-螺旋107-114移动了约1.5 π，破坏了Thr 109的骨架羰基和Gly 113的骨架酰胺基之间的氢键。这种构象的变化是没有预料到的设计proceeding.Compensating相互作用的幅度高达1.1千卡/摩尔发生的一些组的突变，而其他集显示几乎加性的稳定性变化。在实验误差范围内，双突变体V111 F/F153 L的稳定性是相加的，ΔΔ G与两个单突变体的总和仅相差0.1 kcal/mol。四重突变体L99 F/M102 L/V111 I/F153 L的去稳定性为0.5 kcal/mol，而四个单突变体之和的ΔΔG=-1.6 kcal/ mol。多个突变体显示出比单独的M102 L或V111 I更小的来自野生型的总体结构变化。结构和稳定性的合作变化可以根据单个和多个突变体之间的特定结构差异来合理化。因此，似乎已经实现了T4溶菌酶疏水核心的真正重新包装，对结构、稳定性和活性的影响最小。
An attempt has been made to design modified core-packing arrangements in bacteriophage T4 lysozyme. Alternative replacements of the buried residues Leu99, Met102, Val111 and Phe153 were selected using packing calculations and energy minimization. To test the design procedure, a series of multiple mutants was constructed culminating in the replacement L99F/M102L/V111I/F153L. These variants decrease the stability of T4 lysozyme by approximately 0 to 2 kcal/mol. The crystal structures of a number of the variants were determined. In the variant in which Val111 was replaced by Ile, α-helix 107–114 moved by approximately 1·5 Å, breaking the hydrogen bond between the backbone carbonyl group of Thr109 and the backbone amide group of Gly113. This conformational change was not anticipated by the design procedure.Compensating interactions of magnitude up to 1.1 kcal/mol occur for some sets of mutations, while other sets display nearly additive stability changes. Within experimental error, the stability of the double mutant V111F/F153L is additive, withΔΔGdifferent by only 0.1 kcal/mol from the sum of the two single mutants. The quadruple mutant L99F/M102L/V111I/F153L is destabilized by 0·5 kcal/mol, compared toΔΔG= −1·6 kcal/ mol for the sum of the four single mutants. Multiple mutants show smaller overall structural changes from wild-type than M102L or V111I alone. Co-operative changes in structure and stability can be rationalized in terms of specific structural differences between single and multiple mutants. Genuine repacking of the hydrophobic core of T4 lysozyme with minimal effects on structure, stability and activity thus appears to have been achieved.