Koziński M, Garrett-Roe S, Hamm P
Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
J Phys Chem B. 2008 Jun 26;112(25):7645-50. doi: 10.1021/jp8005734. Epub 2008 May 31.
We investigate the sulfhydryl band of cysteines as a new chromophore for two-dimensional IR (2D-IR) studies of the structure and dynamics of proteins. Cysteines can be put at almost any position in a protein by standard methods of site-directed mutagenesis and, hence, have the potential to be an extremely versatile local probe. Although being a very weak absorber in aqueous environment, the sulfhydryl group gets strongly polarized when situated in an alpha-helix inside the hydrophobic core of a protein because of a strong hydrogen bond to the backbone carbonyl group. The extinction coefficient (epsilon=150 M(-1) cm(-1)) then is sufficiently high to perform detailed 2D-IR studies even at low millimolar concentrations. Using porcine (carbonmonoxy)hemoglobin as an example, which contains two such cysteines in its wild-type form, we demonstrate that spectral diffusion deduced from the 2D-IR line shapes reports on the overall-breathing of the corresponding alpha-helix. The vibrational lifetime of the sulfhydryl group (T1 approximately 6 ps) is considerably longer than that of the much more commonly used amide I mode (approximately 1.0 ps), thereby significantly extending the time window in which spectral diffusion processes can be observed. The experiments are accompanied by molecular dynamics simulations revealing a good overall agreement.
我们研究了半胱氨酸的巯基带，将其作为一种新型发色团用于蛋白质结构与动力学的二维红外（2D - IR）研究。通过定点诱变的标准方法，半胱氨酸几乎可以置于蛋白质的任何位置，因此，它有潜力成为一种极其通用的局部探针。尽管巯基在水环境中是非常弱的吸收体，但当它位于蛋白质疏水核心内的α - 螺旋中时，由于与主链羰基形成强氢键，会强烈极化。此时消光系数（ε = 150 M⁻¹ cm⁻¹）足够高，即使在低毫摩尔浓度下也能进行详细的二维红外研究。以猪（一氧化碳）血红蛋白为例，其野生型形式含有两个这样的半胱氨酸，我们证明从二维红外线形推导的光谱扩散反映了相应α - 螺旋的整体呼吸。巯基的振动寿命（T1约为6 ps）比常用得多的酰胺I模式的振动寿命（约1.0 ps）长得多，从而显著扩展了可观察光谱扩散过程的时间窗口。实验还伴随着分子动力学模拟，结果显示总体吻合良好。
