Liang J J, Chakrabarti B
Ophthalmic Research, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Biochem Biophys Res Commun. 1998 May 19;246(2):441-5. doi: 10.1006/bbrc.1998.8640.
The conformation of lens crystallins in vivo or in a highly concentrated solution is not well established. Most studies were carried out in dilute solutions in which protein-protein interaction is minimal. In order to see whether there is conformational change (tertiary and secondary structures) when crystallin solutions are brought to high concentrations, we have performed the following molecular spectroscopic measurements: circular dichroism (CD) and Fourier transform infrared (FTIR). Near-UV CD measurements showed a more than two-fold increase in CD intensity (molar ellipticity) for the total water-soluble (WS) protein from young calf lens nucleus in a highly concentrated solution (> 300 mg/ml in a 0.01-mm cell), when compared with a dilute solution (1000-fold dilution in a 10-mm cell). The individual crystallins in concentrated solutions also showed an increase in CD intensity, but of different magnitude: alpha-crystallin > beta-crystallin > gamma-crystallin. The increased CD indicates that lens crystallins are in a more compact structure in highly concentrated solutions; they likely undergo a transition from a mobile to an immobile state. Change in near-UV CD usually is caused by restricted mobility of aromatic side groups, particularly Trp. The transition involves not only a change in protein tertiary and/or quaternary structure, but also in protein backbone structure. The change of protein backbone structure was drawn from FTIR measurements. FTIR spectra, sensitive to the secondary structure in the amide I region, could be measured for a highly concentrated solution for which far-UV CD measurement is not feasible. The secondary structure that showed prominent change for alpha-crystallin in a highly concentrated solution was beta-conformation: increase in beta-turn with a concomitant decrease of alpha-helix structure.
晶状体晶状体蛋白在体内或高浓度溶液中的构象尚未完全明确。大多数研究是在蛋白质 - 蛋白质相互作用最小的稀溶液中进行的。为了观察晶状体蛋白溶液浓缩至高浓度时是否存在构象变化（三级和二级结构），我们进行了以下分子光谱测量：圆二色性（CD）和傅里叶变换红外光谱（FTIR）。近紫外CD测量表明，与稀溶液（在10 mm比色皿中稀释1000倍）相比，在高浓度溶液（在0.01 mm比色皿中> 300 mg/ml）中，来自幼年小牛晶状体核的总水溶性（WS）蛋白的CD强度（摩尔椭圆率）增加了两倍多。浓缩溶液中的单个晶状体蛋白也显示出CD强度增加，但幅度不同：α - 晶状体蛋白>β - 晶状体蛋白>γ - 晶状体蛋白。CD增加表明晶状体蛋白在高浓度溶液中具有更紧密的结构；它们可能经历从可移动状态到不可移动状态的转变。近紫外CD的变化通常是由芳香族侧链特别是色氨酸的移动受限引起的。这种转变不仅涉及蛋白质三级和/或四级结构的变化，还涉及蛋白质主链结构的变化。蛋白质主链结构的变化来自FTIR测量。对酰胺I区域二级结构敏感的FTIR光谱可用于测量高浓度溶液，而远紫外CD测量在此种溶液中不可行。在高浓度溶液中，α - 晶状体蛋白二级结构发生显著变化的是β - 构象：β - 转角增加，同时α - 螺旋结构减少。
