Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions
Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions
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DOI：
10.1021/bi9526802
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发表时间：
1996-02-27
期刊：
影响因子：
2.9
通讯作者：
Wagner, G
中科院分区：
文献类型：
--
作者：
Lefevre, JF;Dayie, KT;Wagner, G
酵母转录激活子GAL4的DNA结合域（残基1-65）仅部分折叠。当残基10-41 （DNA识别结构域）在游离蛋白中形成明确的结构时，整个多肽在结合DNA时折叠并二聚。为了描述蛋白质的迁移性，我们使用简化的谱密度作图方法（全谱密度作图技术的近似方法）表征了GAL4（1-65）的N-H键向量运动的频谱[Peng， J. W., & Wagner， G, (1992a) J. Magn.。《理性》，98,308-332；彭建伟，王晓明，张晓明，（2009）生物化学学报（自然科学版），31(2):571- 586。在10℃和11.74 t下测定了65个主酰胺基团中的52个基团的N-15自旋-晶格弛豫[R(N)(N-x,N-y)]、自旋-自旋弛豫[R(N)（H-z）—> N-z)]、双自旋序[R(NH)(2H(z))N(x,y))]和反相[R(NH)(2H(z)N(x))]的谱密度函数，用R(N)(N-z) （N-x,N-y）、R（N)（H-z）—> N-z）和R(NH)(2H（z)N(z)）的简化集合计算的谱密度函数与使用所有六组计算的结果非常吻合。简化的方法还有一个额外的优点，即在高场值下看到的错误行为被规避了。在J（ω (N)）和J(0)之间发现了线性相关，J(0)值的有限和明确定义的范围，该范围定义了GAL4（1-65）中内部运动的速率范围。所有残基似乎都经历了两种运动的组合：一种是整体翻滚（相关时间为8.65 ns），另一种是结构的快速内部波动。这些贡献的权重随一级序列的不同而不同，并忠实地反映了蛋白质的二级和三级元素。J（ω (N)）与J(0)在相关线上的位置表示相对于蛋白质整体运动的角度平均量。可以描述内部运动的谱密度函数。
The DNA binding domain (residues 1-65) of the yeast transcriptional activator GAL4 is only partially folded. While residues 10-41, the DNA recognition domain, form a well-defined structure in the free protein, the whole polypeptide folds up and dimerizes upon binding DNA. In order to describe the mobility of the protein, we have characterized the frequency spectrum of the motions of N-H bond vectors of GAL4(1-65) using a reduced spectral density mapping approach (an approximation of the full spectral density mapping technique) [Peng, J. W., & Wagner, G, (1992a) J. Magn. Reson. 98, 308-332; Peng, J. W., & Wagner, G, (1992b) Biochemistry 31, 8571-8586]. N-15 spin-lattice relaxation [R(N)(N-z)], spin-spin relaxation [R(N)(N-x,N-y)], cross-relaxation [R(N)(H-z --> N-z)], two-spin order [R(NH)(2H(z)N(z))], and antiphase [R(NH)(2H(z)N(x,y))] rates were determined for 52 of the 65 backbone amide groups at 10 degrees C anti pH 6.5 at 11.74 T. Calculations of the spectral density functions using a reduced set of R(N)(N-z), R(N)(N-x,N-y), R(N)(H-z --> N-z), and R(NH)(2H(z)N(z)) gave excellent agreement with those calculated using all six sets. The reduced method has the added advantage that the errant behavior seen at high field values is circumvented, A linear correlation was found between J(omega(N)) and J(0) with a limited and clearly defined range of J(0) values which defines the range of rates for internal motions in GAL4(1-65). It appears that all residues experience a combination of two movements: one of the overall tumbling (correlation time, 8.65 ns) and the other of fast internal fluctuations of the structure. The respective weights of these contributions vary with the primary sequence and faithfully mirror the secondary and tertiary elements of the protein. The position on the correlation line of J(omega(N)) versus J(0) indicates the amount of angular averaging relative to the overall motion of the protein. A spectral density function for internal motions can be described.