Garner Dewain K, Vaughan Mark D, Hwang Hee Jung, Savelieff Masha G, Berry Steven M, Honek John F, Lu Yi
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
J Am Chem Soc. 2006 Dec 13;128(49):15608-17. doi: 10.1021/ja062732i.
The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in < 6 nm shifts in the S(Cys)-Cu charge transfer (CT) band in the electronic absorption spectra and < 8 gauss changes in the copper hyperfine coupling constants (AII) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of AII differs, suggesting additional factors influencing AII. In contrast to the small changes in the UV-vis and EPR spectra, a large variation of > 227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.
利用表达蛋白连接技术，将铜绿假单胞菌天青蛋白（Az）中保守的轴向配体甲硫氨酸121替换为同结构的非天然氨基酸类似物，即氧代甲硫氨酸（OxM）、二氟甲硫氨酸（DFM）、三氟甲硫氨酸（TFM）、硒代甲硫氨酸（SeM）和正亮氨酸（Nle）。这些替换导致电子吸收光谱中S（Cys）-Cu电荷转移（CT）带发生小于6 nm的位移，以及X波段电子顺磁共振光谱中铜超精细偶合常数（AII）发生小于8高斯的变化，这表明甲硫氨酸的同结构替换对铜中心的结构扰动最小。CT带的轻微蓝移遵循配体电负性更强的趋势。这一趋势得到了氟化甲硫氨酸类似物的19F NMR研究的支持。然而，AII的顺序不同，表明还有其他因素影响AII。与紫外可见光谱和电子顺磁共振光谱中的微小变化形成对比的是，在此报道的一系列变体的还原电位观察到大于227 mV的巨大变化。此外，还建立了变体的还原电位与疏水性之间的线性相关性。将该分析扩展到其他含1型铜的蛋白质，发现疏水性变化与还原电位变化之间存在线性相关性，与蛋白质支架、实验条件、测量技术和空间修饰无关。该分析还首次揭示了1型中心的高电位和低电位状态，这种差异可能归因于稳定1型中心的疏水或氢键相互作用的破坏导致蛋白质折叠的不稳定。
