Abstract：
The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (μs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) \"locked off\", Fe(II)-CO \"on\", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.
摘要：
转录因子CooA是CRP/FNR(cAMP受体蛋白/富马酸和硝酸还原酶)超家族蛋白,其使用血红素来感测一氧化碳(CO)。CooA响应于CO结合的变构激活目前被描述为一系列离散的结构变化。没有过多考虑蛋白质动力学在DNA结合过程中的潜在作用。这项工作使用定点自旋标记电子顺磁共振波谱（SDSL-EPR）来探测具有氧化还原稳定的氮氧化物自旋标记的CooA的慢时间尺度（μs-ms）构象动力学，和红外光谱来探测CO结合血红素的环境。创建了一系列半胱氨酸取代变体，以在关键功能区域选择性标记CooA，血红素结合域，4/5循环，铰链区，和DNA结合域。在三种功能状态下比较了标记的CooA变体的EPR光谱：Fe（III）“锁定”，Fe(II)-CO“on”，Fe(II)-CO与DNA结合。我们观察到多组分EPR光谱在每个位置的变化;最值得注意的是在铰链区和DNA结合域,扩大对CooA变构机制的描述，以包括蛋白质动力学在DNA结合中的作用。IR振动频率和谱带展宽的DNA依赖性变化进一步表明，活性WT蛋白存在构象异质性，并且DNA结合改变了血红素结合CO的环境。
