Yutani K, Akutsu H, Ogasahara K, Tsujita T, Kyogoku Y
Institute for Protein Research, Osaka University, Japan.
Biochemistry. 1987 Sep 8;26(18):5666-71. doi: 10.1021/bi00392a013.
In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs. Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92. The replacement did not affect the conformation of the protein significantly. The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues. On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH. NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another. The present results are discussed in the light of the conformational stability of the protein.
为了阐明单个氨基酸取代对溶液中大肠杆菌色氨酸合酶α亚基构象的影响，在不同pH值下测量了野生型和突变型蛋白质的1H NMR光谱。通过使用将苏氨酸取代92位组氨酸的突变蛋白，将α亚基四个组氨酸C2质子共振中的两个归属于92位和146位的两个组氨酸残基。这种取代对蛋白质的构象没有显著影响。利用在所有苯丙氨酸残基处氘代的野生型α亚基，可以从其他共振峰中分辨出芳香区所有酪氨酸残基的质子共振。通过比较野生型蛋白质与49位谷氨酸被甲硫氨酸取代的突变型蛋白质的光谱，得出结论：在酸性pH下，这种取代仅影响靠近被取代残基的残基，但在碱性pH下，蛋白质的更大一部分会受到影响。NOE实验表明，五个酪氨酸残基彼此靠近，其中四个位于49位附近。根据蛋白质的构象稳定性对目前的结果进行了讨论。
