Probing protein conformation with a minimal photochemical reagent
Probing protein conformation with a minimal photochemical reagent
复制标题
DOI：
10.1110/ps.4710102
复制
发表时间：
2002-06-01
期刊：
影响因子：
8
通讯作者：
Delfino, JM
中科院分区：
文献类型：
--
作者：
Craig, PO;Ureta, DB;Delfino, JM
H-3-DZN是一种大小与水相似的光反应气体，用于探测蛋白质表面和内部空间的形貌。3 H-DZN光解生成H-3-亚甲基卡宾，其与其分子笼发生非选择性反应，甚至插入C-H键。用1 mM H-3-DZN标记牛α-乳白蛋白（α-LA，MW：14，200）产生0.0041 mol CH 2/mol蛋白质，与预期的非特异性表面标记现象一致。合作尿素诱导展开的α-LA，监测的H-3-亚甲基标记的程度，同意在远和近紫外区域的圆二色光谱测量。在8 M尿素下，未折叠状态U被标记为比天然状态N多25-30%，主要是因为在未折叠时发生的蛋白质的可及表面积（阿萨）的增加。然而，这一结果低于从美国州阿萨的理论估计预期的类似于100%的增量。其中，其他因素，最有可能存在的残余结构，在U中，涉及螺旋H2和H4的亚结构域，可能会占这一事实，如所示的肽标记模式的N和U样品的比较分析。在本文中，我们证明了H-3-亚甲基标记方法的有用性，以监测构象转变和映射溶剂可及性沿着多肽序列，从而打开了概述规范状态的结构特征的可能性（即，变性状态、熔融球）。我们预计，这种技术也将有助于确定蛋白质中的配体结合和寡聚化位点。
H-3-diazirine (H-3-DZN), a photoreactive gas similar in size to water, was used to probe the topography of the surface and inner space of proteins. On photolysis 3H-DZN generates H-3-methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds. Labeling of bovine alpha-lactalbumin (alpha-LA, MW: 14,200) with 1 mM H-3-DZN yielded 0.0041 mol CH2/mol of protein, in agreement with the expectation for an unspecific surface-labeling phenomenon. The cooperative urea-induced unfolding of alpha-LA, as monitored by the extent of H-3-methylene labeling, agrees with that measured by circular dichroism spectroscopy in the far and near ultraviolet regions. At 8 M urea, the unfolded state U was labeled 25-30% more than the native state N primarily because of the increase in the accessible surface area (ASA) of the protein occurring upon unfolding. However, this result lies below the similar to100% increment expected from theoretical estimates of ASA of state U. Among, other factors, most likely the existence of a residual structure in U, that involves helices H2 and H4 of the a subdomain, might account for this fact, as shown by a comparative analysis of peptide labeling patterns of N and U samples. In this paper, we demonstrate the usefulness of the H-3-methylene labeling method to monitor conformational transitions and map solvent accessibility along the polypeptide sequence, thus opening the possibility of outlining structural features of normative states (i.e., denatured states, molten globule). We anticipate that this technique also would help to identify ligand binding and oligomerization sites in proteins.