Eun Ye-Jin, Kurt Neşe, Sekhar Ashok, Cavagnero Silvia
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
J Mol Biol. 2008 Feb 22;376(3):879-97. doi: 10.1016/j.jmb.2007.11.038. Epub 2007 Nov 22.
Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent Delta G degrees (UN(w))=-3.1+/-0.3 kcal mol(-1); m(UN)=-1.7 kcal mol(-1) M(-1)] and predominant alpha-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (beta(I)=0.9+/-0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.
尽管珠蛋白折叠在大多数生物中广泛存在，但迄今为止，只有真核生物珠蛋白被用作折叠/稳定性研究的模型蛋白。这项工作首次对原核生物珠蛋白进行了热力学和动力学表征，即来自大肠杆菌的黄素血红蛋白（apoHmpH）血红素结合结构域的脱辅基形式。这种细菌珠蛋白与真核类似物相比，序列差异很大，但结构几乎相同。我们表明，apoHmpH是一种折叠良好的单体蛋白，在室温下具有中等稳定性[表观ΔG°（UN(w)）=-3.1±0.3 kcal mol⁻¹；m(UN)=-1.7 kcal mol⁻¹ M⁻¹]，且主要为α螺旋结构。值得注意的是，apoHmpH是迄今为止已知折叠速度最快的珠蛋白，其重新折叠速度比真核类似物（如抹香鲸肌红蛋白和大豆豆血红蛋白）快约4至16倍，形成一个具有显著螺旋含量的紧密动力学中间体（β(I)=0.9±0.2）。此外，单个色氨酸Trp120（位于天然H螺旋中）在6毫秒内就锁定在完全类似天然的环境中，这表明该残基及其最接近的空间邻居以超快（亚毫秒）速度完成折叠。总之，apoHmpH是一种细菌珠蛋白，它与其真核类似物具有相同的一般折叠模式（即快速爆发阶段后接较慢的速率决定阶段），但整体折叠速度更快，且动力学中间体具有一些类似天然的特征。这项研究支持了这样一种观点，即细菌和真核生物珠蛋白的一般折叠特征在进化过程中得以保留，而动力学细节则有所不同。
