Exploring protein interfaces with a general photochemical reagent
Exploring protein interfaces with a general photochemical reagent
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DOI：
10.1110/ps.051960406
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发表时间：
2006-04-01
期刊：
影响因子：
8
通讯作者：
Delfino, JM
中科院分区：
文献类型：
--
作者：
Gómez, GE;Cauerhff, A;Delfino, JM
蛋白质折叠、自然构象变化或参与识别现象的伴侣之间的相互作用导致多肽链的溶剂可及表面积（SASA）的差异。该初级事件可以通过沿着蛋白质序列的官能团的不同化学反应性来监测。二氮丙啶（DZN）是一种大小与水相似的光反应气体，可生成亚甲基卡宾（：CH 2）。这种物种的极端化学反应性允许其直接分子笼几乎瞬间和不加选择地修改。H-3-DZN在本实验室成功地用于蛋白质结构和折叠的研究。在这里，我们第一次解决这个探针的有用性，以检查蛋白质-蛋白质复合物中的相互作用的区域。为此，我们选择了鸡蛋白色溶菌酶（HEWL）和单克隆抗体IgG（1）D1.3之间形成的复合物。游离HEWL或与IgG 1 D1.3复合的CH 2标记在1 mM DZN浓度下分别产生2.76和2.32 mmol CH 2/mol蛋白质。这种减少（15%）与从晶体学数据（11%）得出的络合后发生的HEWL的SASA的预期减少一致，与已知的非特异性表面标记反应CH 2一致。胰蛋白酶肽水平的进一步比较分析导致了相互作用中所涉及的位点的鉴定。值得注意的是，与接触区域相关的肽显示出最高的差异标记：H（15）GLDNYR（21）、G（117）TDVQAWIR（125）和G（22）YSLGNWVCAAK（33）。因此，蛋白质足迹与DZN出现作为一个可行的方法，用于映射大分子组装体中涉及的蛋白质结构域的接触区域。
Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. H-3-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme(HEWL) and the monoclonal antibody IgG(1) D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of : CH2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H(15)GLDNYR(21), G(117)TDVQAWIR(125), and G(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.