Munro I, Pecht I, Stryer L
Proc Natl Acad Sci U S A. 1979 Jan;76(1):56-60. doi: 10.1073/pnas.76.1.56.
The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (varphi) of 9.9 nsec at 20 degrees C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits varphi of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8 degrees C (varphi = 31.4 nsec), whereas at 43 degrees C it rotates rapidly (varphi(1) = 0.14 nsec) within a cone of semiangle 26 degrees in addition to rotating together with the whole protein (varphi(2) = 14 nsec). Of particular interest in the large angular range (semiangle, 34 degrees ) and fast rate (varphi(1) = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.
通过时间分辨荧光偏振光谱研究了蛋白质分子在亚纳秒和纳秒时间范围内的动力学。来自储存环的同步辐射用作脉冲光源，以激发一系列蛋白质中的单个色氨酸残基。检测到的光脉冲的半高宽为0.65纳秒，这使得在亚纳秒时间范围内测量发射各向异性动力学并从而解析内部旋转运动成为可能。所研究的蛋白质在亚纳秒时间范围内其色氨酸残基表现出不同程度的旋转自由度，范围从几乎没有移动性到几乎完全自由。金黄色葡萄球菌核酸酶B（20,000道尔顿）的色氨酸残基在20℃下具有9.9纳秒的单一旋转相关时间（φ），对应于整个蛋白质分子的旋转。相比之下，牛碱性A1髓磷脂蛋白（18,000道尔顿）表现出0.09和1.26纳秒的φ，表明该蛋白质中的色氨酸残基具有高度灵活性。人血清白蛋白（69,000道尔顿）的单个色氨酸在8℃时几乎没有旋转自由度（φ = 31.4纳秒），而在43℃时，它除了与整个蛋白质一起旋转（φ(2) = 14纳秒）之外，还在半角为26度的圆锥体内快速旋转（φ(1) = 0.14纳秒）。特别值得关注的是铜绿假单胞菌天青蛋白（14,000道尔顿）中色氨酸残基的大角度范围（半角，为34度）和快速旋转速率（φ(1) = 0.51纳秒）。已知该残基位于蛋白质的疏水内部。观察到的这些色氨酸残基内部运动的幅度和速率表明，在功能上重要的构象变化中的基本步骤可能发生在亚纳秒时间范围内。
