Lefevre J F, Dayie K T, Peng J W, Wagner G
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1996 Feb 27;35(8):2674-86. doi: 10.1021/bi9526802.
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]. 15N spin-lattice relaxation [Rn(Nz)], spin-spin relaxation [Rn(Nx,y)], cross-relaxation [RN(Hz-->Nz)], two-spin order [RNH(2HzNz)], and antiphase [RNH(2HzNx,y)] rates were determined for 52 of the 65 backbone amide groups at 10 degrees C and ph 6.5 at 11.74 T. Calculations of the spectral density functions using a reduced set of RN(Nz),RN(Nx,y),RN(Hz-->Nz), and RNH(2HzNz) 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.
酵母转录激活因子GAL4的DNA结合结构域（第1至65位氨基酸残基）仅部分折叠。虽然第10至41位氨基酸残基构成的DNA识别结构域在游离蛋白中形成了明确的结构，但整个多肽在结合DNA时会折叠并二聚化。为了描述该蛋白的运动性，我们使用简化谱密度映射方法（全谱密度映射技术的一种近似方法）[Peng, J. W., & Wagner, G. (1992a) J. Magn. Reson. 98, 308 - 332; Peng. J. W., & Wagner, G. (1992b) Biochemistry 31, 8571 - 8586]对GAL4(1 - 65)的N - H键向量运动的频谱进行了表征。在10℃、pH 6.5以及11.74 T的条件下，测定了65个主链酰胺基团中52个的15N自旋晶格弛豫速率[Rn(Nz)]、自旋 - 自旋弛豫速率[Rn(Nx,y)]、交叉弛豫速率[RN(Hz→Nz)]、双自旋序[RNH(2HzNz)]以及反相[RNH(2HzNx,y)]。使用简化的Rn(Nz)、Rn(Nx,y)、RN(Hz→Nz)和RNH(2HzNz)集合计算谱密度函数，与使用所有六个集合计算得到的结果高度吻合。简化方法的额外优势在于避免了在高场值时出现的异常行为。发现J(ωN)与J(0)之间存在线性相关性，且J(0)值的范围有限且明确，该范围定义了GAL4(1 - 65)内部运动的速率范围。似乎所有残基都经历了两种运动的组合：一种是整体翻滚运动（相关时间为8.65 ns），另一种是结构的快速内部波动。这些贡献的各自权重随一级序列而变化，并忠实地反映了蛋白质的二级和三级结构元件。J(ωN)相对于J(0)在相关线上的位置表明了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。
