A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor
A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor
复制标题
一种量化酶的活性如何受其最近拥挤邻居的净电荷影响的方法
DOI：
10.1002/pro.4384
复制
发表时间：
2022-08-11
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白质拥挤的静电效应尚未得到系统的研究。相反，蛋白质拥挤通常使用静电中性的共溶剂或拥挤剂进行研究，没有方法测量拥挤剂的净电荷（Z）如何影响蛋白质功能。例如，在拥挤的环境中，酶的活性是否会受到其邻居的净电荷的静电影响？本文报道了一种通过半随机化学交联将不同净电荷的蛋白质聚集到酶上的方法。作为概念证明，RNA酶A通过与不同的血红素蛋白交联而聚集（在≤德拜长度的距离处），其中Z = +8.50 ± 0.04，Z = +6.39 ± 0.12或Z =-10.30 ± 1.32。交联没有破坏蛋白质的结构，根据酰胺H/D交换，并没有抑制RNA酶A的活性。对于RNase A，我们发现每个拥挤邻居的静电环境对RNA水解速率有显著影响。拥挤与阳离子细胞色素c导致的活动增加，而拥挤与阴离子“增压”细胞色素c或肌红蛋白减少活动。令人惊讶的是，静电拥挤效应在高离子强度（I = 0.201 M）下被放大，在低离子强度（I = 0.011 M）下被衰减。这种盐依赖性可能是由二聚体间隙处的一组独特的双电层引起的（最大距离为8 μ m，不能容纳四层）。这种通过交联进行拥挤的新方法可用于研究蛋白质拥挤中的静电效应。
The electrostatic effects of protein crowding have not been systematically explored. Rather, protein crowding is generally studied with co‐solvents or crowders that are electrostatically neutral, with no methods to measure how the net charge (Z) of a crowder affects protein function. For example, can the activity of an enzyme be affected electrostatically by the net charge of its neighbor in crowded milieu? This paper reports a method for crowding proteins of different net charge to an enzyme via semi‐random chemical crosslinking. As a proof of concept, RNase A was crowded (at distances ≤ the Debye length) via crosslinking to different heme proteins with Z = +8.50 ± 0.04, Z = +6.39 ± 0.12, or Z = −10.30 ± 1.32. Crosslinking did not disrupt the structure of proteins, according to amide H/D exchange, and did not inhibit RNase A activity. For RNase A, we found that the electrostatic environment of each crowded neighbor had significant effects on rates of RNA hydrolysis. Crowding with cationic cytochrome c led to increases in activity, while crowding with anionic “supercharged” cytochrome c or myoglobin diminished activity. Surprisingly, electrostatic crowding effects were amplified at high ionic strength (I = 0.201 M) and attenuated at low ionic strength (I = 0.011 M). This salt dependence might be caused by a unique set of electric double layers at the dimer interspace (maximum distance of 8 Å, which cannot accommodate four layers). This new method of crowding via crosslinking can be used to search for electrostatic effects in protein crowding.
登录
查看更多内容
影响因子：
16.6
作者：
Fried SD;Boxer SG
通讯作者：
Boxer SG
影响因子：
7.4
作者：
Abdolvahabi, Alireza;Gober, Jennifer L.;Shaw, Bryan F.
通讯作者：
Shaw, Bryan F.
DOI：
10.1093/protein/14.12.983
发表时间：
2001-12-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
James, DA;Burns, DC;Woolley, GA
通讯作者：
Woolley, GA
影响因子：
2.9
作者：
Cuchillo, Claudi M.;Victoria Nogues, M.;Raines, Ronald T.
通讯作者：
Raines, Ronald T.
DOI：
10.1021/jp506594f
发表时间：
2014-09-11
期刊：
The journal of physical chemistry. B
影响因子：
--
作者：
Aumiller WM Jr;Davis BW;Hatzakis E;Keating CD
通讯作者：
Keating CD