Initiation sites of protein folding by NMR analysis.

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable. IMAGES:

精准表征蛋白质变性态，对于阐明引导海量可能构象经由快速路径完成折叠的相互作用机制至关重要。基于核奥弗豪泽效应（nuclear Overhauser effect，NOE）的核磁共振（nuclear magnetic resonance，NMR）实验可检测空间邻近的氢原子，并提供蛋白质局部结构的相关信息。本研究开发了一种NMR实验方法，可检测尿素溶液中蛋白质巴纳酶（barnase）酰胺氢原子间几乎所有的序列相邻NOEs（即HN-HN NOE），涵盖其无规卷曲区域的相关信号。通过对这些HN-HN NOE进行半定量分析，我们鉴定出了部分结构化区域，该结果与折叠通路早期形成的结构区域高度吻合。本研究结果强烈表明，巴纳酶的折叠起始于第一个α螺旋以及第三与第四股β折叠之间的β转角。这种鉴定残余结构的策略同样适用于冷变性巴司塔酶（barstar）以及盐酸胍变性的胰凝乳蛋白酶抑制剂2（chymotrypsin inhibitor 2），因此该方法具有广泛的适用性。IMAGES: