Buck M, Radford S E, Dobson C M
Oxford Centre for Molecular Sciences, University of Oxford, England.
Biochemistry. 1993 Jan 19;32(2):669-78. doi: 10.1021/bi00053a036.
The effect of 2,2,2-trifluoroethanol (TFE) on the solution conformation of hen egg white lysozyme has been investigated using circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. Addition of TFE to lysozyme at pH 2.0, 27 degrees C, up to a concentration of 15% (v/v) induces only slight changes in the NMR spectrum. However, above this concentration a cooperative transition to a new but partially structured state of the protein is observed. This state shows no structural cooperativity against further denaturation and is characterized by an ellipticity in the far-UV CD greater than that of the native protein. Near-UV CD intensity is dramatically reduced compared with that of the native state, and 1H NMR studies indicate that side-chain interactions are substantially averaged in this denatured state. Solvent proton/deuterium exchange rates for 66 amide hydrogens were measured site-specifically by a combination of amide trapping experiments and 2D 1H NMR. Significant protection from exchange occurs for about 25 backbone amides, the majority of which are located in regions of the protein that are helical in the native enzyme. By contrast, amides located in a second region of the native protein which contains a beta-sheet and one 3(10)-helix as well as a long loop show little protection. This pattern of protection resembles that found in the stable molten globule state of alpha-lactalbumin and in an early kinetic intermediate detected in the refolding of hen lysozyme.
利用圆二色性（CD）和核磁共振氢谱（1H NMR）研究了2,2,2-三氟乙醇（TFE）对鸡蛋清溶菌酶溶液构象的影响。在pH 2.0、27℃条件下，向溶菌酶中添加TFE至浓度达15%（v/v）时，NMR谱仅出现轻微变化。然而，高于此浓度时，可观察到蛋白质协同转变为一种新的但部分结构化的状态。该状态对进一步变性无结构协同性，其远紫外CD椭圆率大于天然蛋白质。与天然状态相比，近紫外CD强度显著降低，1H NMR研究表明在这种变性状态下侧链相互作用基本平均化。通过酰胺捕获实验和二维1H NMR相结合的方法，位点特异性地测定了66个酰胺氢的溶剂质子/氘交换率。约25个主链酰胺有显著的交换保护作用，其中大部分位于天然酶中呈螺旋结构的蛋白质区域。相比之下，位于天然蛋白质第二个区域（包含一个β-折叠、一个3(10)-螺旋以及一个长环）的酰胺几乎没有保护作用。这种保护模式类似于在α-乳白蛋白的稳定熔球状态以及在鸡蛋清溶菌酶重折叠过程中检测到的早期动力学中间体中发现的模式。
