King S C, Hansen C L, Wilson T H
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, MA 02115.
Biochim Biophys Acta. 1991 Feb 25;1062(2):177-86. doi: 10.1016/0005-2736(91)90390-t.
The lacY from Escherichia coli strains 020 and AE43 have been cloned on plasmids which were designated p020-K358T and pAE43-D237N. These lacY mutants contain amino acid substitutions changing Lys-358 to Thr or Asp-237 to Asn, respectively. The charge neutralizing effect of each mutation is associated with a functional defect in melibiose transport which we exploited in order to isolate second site revertants to the melibiose-positive phenotype. Eleven melibiose-positive revertants of p020-K358T were isolated. All contained a second-site mutation converting Asp-237 to a neutral amino acid (8 to Asn, 1 to Gly, and 2 to Tyr). Twelve melibiose-positive revertants of pAE43-D237N were isolated. Two were second-site revertants converting Lys-358 to a neutrally Gln residue, while the remainder directly reverted Asn-237 to the wild-type Asp-237. We conclude that the functional intimate relationship between Asp-237 and Lys-358 suggests that these residues may be closely juxtaposed in three-dimensional space, possibly forming a 'charge-neutralizing' salt bridge.
来自大肠杆菌菌株020和AE43的lacY已被克隆到分别命名为p020 - K358T和pAE43 - D237N的质粒上。这些lacY突变体分别含有将赖氨酸-358变为苏氨酸或天冬氨酸-237变为天冬酰胺的氨基酸替换。每个突变的电荷中和效应与蜜二糖转运的功能缺陷相关，我们利用这一缺陷来分离出向蜜二糖阳性表型回复的第二位点回复突变体。分离出了11个p020 - K358T的蜜二糖阳性回复突变体。所有回复突变体都含有一个将天冬氨酸-237转变为中性氨基酸的第二位点突变（8个变为天冬酰胺，1个变为甘氨酸，2个变为酪氨酸）。分离出了12个pAE43 - D237N的蜜二糖阳性回复突变体。其中2个是将赖氨酸-358转变为中性谷氨酰胺残基的第二位点回复突变体，其余的则直接将天冬酰胺-237回复为野生型天冬氨酸-237。我们得出结论，天冬氨酸-237和赖氨酸-358之间的功能密切关系表明，这些残基在三维空间中可能紧密相邻，可能形成一个“电荷中和”盐桥。
