Dynamic interchanging native states of lymphotactin examined by SNAPP-MS.
Dynamic interchanging native states of lymphotactin examined by SNAPP-MS.
复制标题
通过 SNAPP-MS 检查淋巴趋化素的动态交换天然状态。
DOI：
10.1007/s13361-010-0042-3
复制
发表时间：
2011
影响因子：
3.2
通讯作者：
Julian,RyanR
中科院分区：
文献类型：
--
作者：
Sun,Qingyu;Tyler,RobertC;Volkman,BrianF;Julian,RyanR
人类趋化因子趋化蛋白（Ltn）是一种引人注目的蛋白质，在凝聚相中在两个不相关的天然状态结构之间相互转换。有可能通过选择的序列取代将平衡向任一构象移动。先前的结果表明，二硫键稳定的变体优先采用典型的趋化因子折叠（Ltn 10），而单个氨基酸的变化（W55 D）有利于新的Ltn 40二聚体结构。选择性非共价加合物蛋白质探针法（SNAPP）是近年来发展起来的一种检测液相蛋白质结构的方法。在本文中，它表明SNAPP可以很容易地识别和区分Ltn 10和Ltn 40状态的光甲素在水溶液中。还使用SNAPP检查了CC 3、W55 D和野生型蛋白质的有机变性剂、酸和二硫键还原和封闭的影响。只有二硫键还原显着扰动的蛋白质，并导致在相当大的减少加合物的形成与损失的三级/二级结构一致。冷变性实验表明，野生型Ltn是三种蛋白质中对温度最敏感的。在所有实验中的较高电荷状态的检查，这被假定为代表Ltn-10和Ltn-40之间的过渡态结构，揭示了增加的18 C6连接相对于更多的折叠结构。这一观察结果与竞争性分子内氢键的增加是一致的，这可能会引导过渡。研究气相结构的实验表明，所有三种蛋白质在气相中可以在结构上区分。此外，气相实验能够识别优选的加合物结合位点。
The human chemokine lymphotactin (Ltn) is a remarkable protein that interconverts between two unrelated native state structures in the condensed phase. It is possible to shift the equilibrium toward either conformation with selected sequence substitutions. Previous results have shown that a disulfide-stabilized variant preferentially adopts the canonical chemokine fold (Ltn10), while a single amino acid change (W55D) favors the novel Ltn40 dimeric structure. Selective noncovalent adduct protein probing (SNAPP) is a recently developed method for examining solution phase protein structure. Herein, it is demonstrated that SNAPP can easily recognize and distinguish between the Ltn10 and Ltn40 states of lymphotactin in aqueous solution. The effects of organic denaturants, acid, and disulfide bond reduction and blocking were also examined using SNAPP for the CC3, W55D, and wild type proteins. Only disulfide reduction was shown to significantly perturb the protein, and resulted in considerably decreased adduct formation consistent with loss of tertiary/secondary structure. Cold denaturation experiments demonstrated that wild-type Ltn is the most temperature sensitive of the three proteins. Examination of the higher charge states in all experiments, which are presumed to represent transition state structures between Ltn-10 and Ltn-40, reveals increased 18C6 attachment relative to the more folded structures. This observation is consistent with increased competitive intramolecular hydrogen bonding, which may guide the transition. Experiments examining the gas phase structures revealed that all three proteins can be structurally distinguished in the gas phase. In addition, the gas phase experiments enabled identification of preferred adduct binding sites.