Wilson G, Ford S J, Cooper A, Hecht L, Wen Z Q, Barron L D
Chemistry Department, The University, Glasgow, UK.
J Mol Biol. 1995 Dec 8;254(4):747-60. doi: 10.1006/jmbi.1995.0652.
Proteins in aqueous solution are now accessible to Raman optical activity (ROA) measurements, which provide an incisive new probe of secondary and tertiary structure illustrated here by a study of bovine alpha-lactalbumin. The room-temperature ROA spectrum of native bovine alpha-lactalbumin is similar to that of native hen egg-white lysozyme except for features attributable to differences in the loop regions: in particular, a positive ROA band at approximately 1338 cm-1 assigned to conformationally homogeneous loop structure, possibly with local order corresponding to 3(10)-helix, has more than double the intensity in alpha-lactalbumin compared with lysozyme. This is consistent with the two proteins having similar secondary structure but different local details in the tertiary fold. ROA measurements on alpha-lactalbumin at pH 2.0 over a range of temperatures have provided a new perspective on the molten globule state. Thus at 35 degrees C ROA reveals the presence of some secondary structure but an almost complete loss of the tertiary loop structure; whereas at 2 degrees C the ROA spectrum is almost identical with that of the native protein, which is strong evidence that virtually all of the secondary structure and the tertiary backbone fold persist, albeit within a looser framework associated with increased solvent exposure and change of environment of many of the side-chains as evidenced by an increase in noise and bandwidth of some of the ROA signals together with aromatic fluorescence and near-UV circular dichroism signals characteristic of the molten globule state. Our sample of acid alpha-lactalbumin at 2 degrees C therefore appears to be an archetypal example of Ptitsyn's "native-like" molten globule, having a fixed native-like tertiary fold but with loss of tight packing of the side-chains; whereas at 35 degrees C it is a "disordered" molten globule. At 20 degrees C the acid molten globule appears to retain highly native-like secondary structure but with most of the tertiary fold already lost. A calcium-free sample of alpha-lactalbumin at neutral pH displayed a broad cooperative transition between native and molten globule states at approximately 15 degrees C, with the latter state showing similar but somewhat degraded tertiary loop ROA signatures to the native protein. In both the acid and apo molten globule states the ROA signatures of the secondary structure and the tertiary loops showed a gradual change with temperature.
水溶液中的蛋白质现在可以进行拉曼光学活性（ROA）测量，这为二级和三级结构提供了一种敏锐的新探针，本文通过对牛α-乳白蛋白的研究进行了说明。天然牛α-乳白蛋白的室温ROA光谱与天然鸡蛋清溶菌酶的光谱相似，除了归因于环区差异的特征：特别是，在约1338 cm-1处的一个正ROA带被指定为构象均匀的环结构，可能具有对应于3(10)-螺旋的局部有序，与溶菌酶相比，α-乳白蛋白中的强度增加了一倍多。这与这两种蛋白质具有相似的二级结构但三级折叠中的局部细节不同是一致的。在一系列温度下对pH 2.0的α-乳白蛋白进行的ROA测量为熔球态提供了新的视角。因此，在35℃时，ROA显示存在一些二级结构，但三级环结构几乎完全丧失；而在2℃时，ROA光谱与天然蛋白质的光谱几乎相同，这有力地证明几乎所有的二级结构和三级主链折叠都得以保留，尽管是在一个与溶剂暴露增加和许多侧链环境变化相关的较宽松框架内，这由一些ROA信号的噪声和带宽增加以及熔球态特有的芳香族荧光和近紫外圆二色性信号所证明。因此，我们在2℃下的酸性α-乳白蛋白样品似乎是普季茨yn的“类天然”熔球的典型例子，具有固定的类天然三级折叠但侧链紧密堆积丧失；而在35℃时它是一个“无序”熔球。在20℃时，酸性熔球似乎保留了高度类天然的二级结构，但大部分三级折叠已经丧失。中性pH下的无钙α-乳白蛋白样品在约15℃时显示出天然态和熔球态之间的广泛协同转变，后者状态显示出与天然蛋白质相似但略有降解的三级环ROA特征。在酸性和脱辅基熔球态下，二级结构和三级环的ROA特征都随温度逐渐变化。
