Moss J A, Silinski P, Fitzgerald M C
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Fresenius J Anal Chem. 2001 Feb;369(3-4):252-7. doi: 10.1007/s002160000644.
A novel mass spectrometry- and chemical synthesis-based approach for studying protein folding reactions is described, and its initial application to study the folding/unfolding reaction of a homo-hexameric enzyme 4-oxalocrotonate (4OT) is reported. This new approach involves the application of total chemical synthesis to prepare protein analogues that contain a photoreactive amino acid site-specifically incorporated into their primary amino acid sequence. To this end, a photoreactive amino acid-containing analogue of 4OT in which Pro-1 was replaced with p-benzoyl-l-phenylalanine (Bpa) was prepared. This analogue can be used to map structurally specific protein-protein interactions in 4OT's native folded state. These photocrosslinking studies and peptide mapping results with (PlBpa)4OT indicate that this construct is potentially useful for probing the structural properties of equilibrium and kinetic intermediates in 4OT's folding reaction.
本文描述了一种基于质谱和化学合成的研究蛋白质折叠反应的新方法，并报道了其在研究同六聚体酶4-草酰巴豆酸（4OT）折叠/去折叠反应中的初步应用。这种新方法涉及应用全化学合成来制备蛋白质类似物，这些类似物含有特异性地掺入其一级氨基酸序列中的光反应性氨基酸。为此，制备了一种4OT的含光反应性氨基酸的类似物，其中Pro-1被对苯甲酰基-L-苯丙氨酸（Bpa）取代。该类似物可用于绘制4OT天然折叠状态下结构特异性的蛋白质-蛋白质相互作用。这些光交联研究以及（PlBpa）4OT的肽图谱结果表明，该构建体可能有助于探究4OT折叠反应中平衡和动力学中间体的结构性质。
