Engineering a Conformationally Switchable Artificial Metalloprotein.
Engineering a Conformationally Switchable Artificial Metalloprotein.
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DOI：
10.1021/jacs.2c08885
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发表时间：
2022-11-30
影响因子：
15
通讯作者：
Olshansky, Lisa
中科院分区：
文献类型：
--
作者：
Fatima, Saman;Boggs, David G.;Ali, Noor;Thompson, Peter J.;Thielges, Megan C.;Bridwell-Rabb, Jennifer;Olshansky, Lisa
许多天然存在的金属酶门控限速构象变化，并存在着一个关键的蛋白质和亚原子的变化影响嵌入的金属辅因子的电子结构的宏观结构重排之间的相互作用。尽管有这种联系，大多数人工金属蛋白（ArMs）是在结构刚性的蛋白质宿主中制备的。为了更好地模拟金属蛋白反应性的自然机制，我们已经开发了构象可切换的ARM（swArMs），进行大规模的结构重排后变构效应结合。本文报道的swArMs含有Co（dmgH）2（X）辅因子（dmgH =丁二酮肟，X = N3−、H3C−和iPr−）。我们使用紫外-可见吸收和能量色散X-射线荧光光谱，沿着蛋白质测定和质谱，以表明这些金属辅因子通过大肠杆菌谷氨酰胺结合蛋白（GlnBP）内的直接Co-S半胱氨酸连接位点特异性和化学计量地安装。通过单晶X射线衍射的结构表征揭示了金属辅因子在蛋白质折叠内的精确定位和微环境。荧光光谱、圆二色光谱、红外光谱、沿着等温滴定量热分析表明，谷氨酰胺的变构结合驱动了大规模的蛋白质构象变化。在swArMs含有Co（dmgH）2（CH 3）辅因子，我们表明，蛋白质稳定，否则不稳定的Co-S键相对于自由复合物。作为温度和pH值的函数进行的动力学研究表明，蛋白质的构象变化加速这种键的解离在pH值依赖的方式。我们目前的swArMs作为一个强大的平台，调查变构和金属辅因子调节之间的相互作用。
Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3−, H3C−, and iPr−). We used UV–vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co–S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co–S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.
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影响因子：
2.9
作者：
Bermejo, Guillermo A.;Strub, Marie-Paule;Ho, Chien;Tjandra, Nico
通讯作者：
Tjandra, Nico
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
5.6
作者：
Hsiao, CD;Sun, YJ;Wang, BC
通讯作者：
Wang, BC
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH