Li Y C, Montelione G T
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854-5638.
Biochemistry. 1995 Feb 28;34(8):2408-23. doi: 10.1021/bi00008a003.
Human type-alpha transforming growth factor (hTGF alpha) is a small mitogenic protein containing 50 amino acids and three disulfide bonds. It has both sequence and structural homology with epidermal growth factor (EGF). While the three-dimensional structures of hTGF alpha and other EGF-like proteins have been studied extensively, relatively little is known about conformational dynamics of these molecules. In this paper we describe nuclear relaxation measurements which probe the molecular dynamics of hTGF alpha in aqueous solution at neutral pH. In order to characterize conformational dynamics of hTGF alpha on both the fast (i.e., sub-nanosecond) and intermediate nitrogen-15 chemical-exchange (i.e., microsecond) time scales, we measured nitrogen-15 relaxation parameters at pH 7.1 +/- 0.1 and a temperature of 30 +/- 0.5 degrees C. Measurements of nitrogen-15 longitudinal (R1) and transverse (R2) relaxation rates, and 1H-15N heteronuclear NOE effects, were then interpreted using an extended Lipari-Szabo analysis [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559; Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., & Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991] to provide estimates of the locations and amplitudes of fast internal motions and the locations of nitrogen-15 chemical-exchange line broadening. These results demonstrate that, under conditions of pH and temperature at which it is tightly bound by the EGF receptor, hTGF alpha is a highly dynamic molecule. Indeed, some 40% of the backbone amide groups of hTGF alpha, including many at the interface between the two subdomains, exhibit significant nitrogen-15 chemical-exchange line broadening indicative of interconversions between multiple protein conformations on the microsecond time scale. The distribution of these sites on the three-dimensional protein structure suggests that these dynamic fluctuations are due to (i) partial unfolding of the core beta-sheet, (ii) hinge-bending motions between the N- and C-terminal subdomains, and/or (iii) disulfide bond isomerization in the solution structure of hTGF alpha at neutral pH.
人α型转化生长因子（hTGFα）是一种含有50个氨基酸和三个二硫键的小分子促有丝分裂蛋白。它与表皮生长因子（EGF）在序列和结构上具有同源性。虽然已经对hTGFα和其他EGF样蛋白的三维结构进行了广泛研究，但对这些分子的构象动力学了解相对较少。在本文中，我们描述了核弛豫测量，该测量用于探测中性pH值下hTGFα在水溶液中的分子动力学。为了在快速（即亚纳秒）和中间氮-15化学交换（即微秒）时间尺度上表征hTGFα的构象动力学，我们在pH 7.1±0.1和温度30±0.5℃下测量了氮-15弛豫参数。然后，使用扩展的Lipari-Szabo分析[Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546 - 4559; Clore, G. M., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., & Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989 - 4991]来解释氮-15纵向（R1）和横向（R2）弛豫率以及1H-15N异核NOE效应的测量结果，以估计快速内部运动的位置和幅度以及氮-15化学交换线展宽的位置。这些结果表明，在被EGF受体紧密结合的pH和温度条件下，hTGFα是一个高度动态的分子。实际上，hTGFα约40%的主链酰胺基团，包括两个亚结构域之间界面处的许多基团，表现出显著的氮-15化学交换线展宽，这表明在微秒时间尺度上多种蛋白质构象之间存在相互转换。这些位点在三维蛋白质结构上的分布表明，这些动态波动是由于（i）核心β折叠的部分展开，（ii）N端和C端亚结构域之间的铰链弯曲运动，和/或（iii）中性pH下hTGFα溶液结构中的二硫键异构化。
