Nowak U K, Li X, Teuten A J, Smith R A, Dobson C M
Oxford Centre for Molecular Sciences, University of Oxford, U.K.
Biochemistry. 1993 Jan 12;32(1):298-309. doi: 10.1021/bi00052a038.
u-PA (urokinase-type plasminogen activator or urokinase) has been studied under a variety of solution conditions by 1-D and 2-D NMR spectroscopy. Very high quality spectra could be obtained from the recombinant protein despite the high molecular mass (46 kDa) by appropriate choice of solution conditions; mildly acidic pH and low ionic strength were found to be optimal. Comparison of spectra of u-PA with spectra of the isolated kringle and protease domains, the EGF-kringle pair, and a synthetic peptide from the kringle-protease linker region, enabled sequential assignments in the u-PA spectrum to be made for kringle resonances, and domain-specific assignments for many others. Simulations of line shapes in both 1-D and 2-D spectra enabled effective correlation times for the different domains, both isolated and in the intact protein, to be determined. These have permitted a model of the u-PA dynamics to be put forward involving extensive, but not unrestricted, motion between the different domains.
已通过一维和二维核磁共振光谱在多种溶液条件下对尿激酶型纤溶酶原激活剂（u-PA）进行了研究。尽管重组蛋白分子量较高（46 kDa），但通过适当选择溶液条件仍可获得高质量的光谱；发现微酸性pH和低离子强度是最佳条件。将u-PA的光谱与分离的kringle结构域和蛋白酶结构域、EGF-kringle对以及kringle-蛋白酶连接区的合成肽的光谱进行比较，能够对u-PA光谱中的kringle共振进行顺序归属，并对许多其他共振进行结构域特异性归属。对一维和二维光谱中的线形进行模拟，可以确定不同结构域（无论是分离的还是完整蛋白中的）的有效相关时间。这些结果使得能够提出一个u-PA动力学模型，该模型涉及不同结构域之间广泛但并非无限制的运动。
