Düx P, Rubinstenn G, Vuister G W, Boelens R, Mulder F A, Hård K, Hoff W D, Kroon A R, Crielaard W, Hellingwerf K J, Kaptein R
Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Biochemistry. 1998 Sep 15;37(37):12689-99. doi: 10.1021/bi9806652.
The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.
嗜盐外硫红螺菌的光感受蛋白——光活性黄色蛋白（PYP）的溶液结构已通过多维核磁共振光谱法测定。该结构由一个开放、扭曲的6股反平行β折叠组成，两侧各有四个α螺旋。最终选定的26个结构对于跨越残基Phe6 - Ala16、Asp24 - Ala112和Tyr118 - Val125的区域定义明确，其主链相对于平均值的均方根偏差为0.45 Å，所有重原子的均方根偏差为0.88 Å。将该溶液结构与早期发表的1.4 Å晶体结构（Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278 - 6287）进行比较，发现定义明确区域的主链均方根偏差为1.77 Å，二者具有相似性。与晶体结构相比，主链最明显的差异出现在N端附近的残基Asp19 - Leu23处，该区域在晶体结构中对应一个α螺旋，而在溶液结构中是定义最不明确的区域之一。为了表征PYP在溶液中的动态行为，我们进行了15N弛豫研究和氢/氘交换测量。通过无模型的Lipari - Szabo方法确定序参数，从而识别出几个动力学增强的区域。对整体结构主链轨迹中的原子位移与核磁共振的迁移率测量结果进行比较，结果表明定义不明确的区域在纳秒到皮秒时间尺度上具有快速的内部运动。
