Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin.
Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin.
复制标题
游离和前结构域复合枯草杆菌蛋白酶中的氢-氘交换。
DOI：
10.1021/bi061601r
复制
发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Bryan,PhilipN
中科院分区：
文献类型：
--
作者：
Sari,Nese;Ruan,Biao;Fisher,KathrynE;Alexander,PatrickA;Orban,John;Bryan,PhilipN
测定了游离和前结构域复合的枯草杆菌蛋白酶中223个酰胺质子的残基特异性交换率，以了解前结构域结合如何影响枯草杆菌蛋白酶折叠的能量学。在游离枯草杆菌蛋白酶中，酰胺质子可根据交换速率分类：74种快速交换剂（速率≥1 h-1）; 52种中等交换剂（速率在1 h-1和1 day-1之间）; 31种慢速交换剂（速率在1 day-1和0.001 day-1之间）。其余66个酰胺蛋白在9个月内没有可检测到的交换（kobs<1年），并表示为核心质子。核心残基出现在枯草杆菌蛋白酶的整个主要结构元件中。前结构域结合导致中心β折叠的高保护因子（100−1000），特别是在β链S5、S6和S7附近以及它们之间的连接环。这些连接环为金属位点B的阳离子提供配体。总的来说，前结构域结合似乎促进了整个中心β折叠和α螺旋C在β链2和3之间的左手交叉连接中的组织。它似乎也有利于异构化的多个脯氨酸后期折叠，使金属网站B的形成。位点B周围稳定区域的增加是以牺牲前结构域结合更远端区域的稳定性为代价的：C-末端α-螺旋H和N-末端α-螺旋A和B。前结构域结合在这些区域中的交换加速揭示了折叠中间体和完整的天然结构之间的拮抗作用。这种拮抗作用有助于解释为什么需要前结构域来稳定折叠中间体，以及为什么游离枯草杆菌蛋白酶的解折叠很少通过该中间体发生。
Residue-specific exchange rates of 223 amide protons in free and prodomain-complexed subtilisin were determined in order to understand how the prodomain binding affects the energetics of subtilisin folding. In free subtilisin, amide protons can be categorized according to exchange rate: 74 fast exchangers (rates ≥1 h-1); 52 medium exchangers (rates between 1 h-1and 1 day-1); 31 slow exchangers (rates between 1 day-1and 0.001 day-1). The remaining 66 amide proteins did not exchange detectibly over 9 months (kobs< year-1) and were denoted as core protons. Core residues occur throughout the main structural elements of subtilisin. Prodomain binding results in high protection factors (100−1000) in the central β-sheet, particularly in the vicinity of β-strands S5, S6, and S7 and the connecting loops between them. These connecting loops provide the ligands to the cation at metal site B. Overall, prodomain binding seems to facilitate the organization of the entire central β-sheet and α-helix C in the left-handed crossover connection between β-strands two and three. It also appears to facilitate the isomerization of multiple prolines late in folding, allowing the formation of metal site B. The gain of stability region around site B comes at the cost of stability in regions more distal to prodomain binding: theC-terminal α-helix H and theN-terminal α-helices A and B. The acceleration of exchange in these regions by prodomain binding reveals an antagonism between the folding intermediate and the full native structure. This antagonism helps to explain why the prodomain is needed to stabilize the folding intermediate as well as why the unfolding of free subtilisin seldom occurs via this intermediate.