Beaulieu C F, Brown R D, Clark J I, Spiller M, Koenig S H
Department of Biological Structure, University of Washington, School of Medicine, Seattle 98195.
Magn Reson Med. 1989 Jun;10(3):362-72. doi: 10.1002/mrm.1910100308.
We have extended our earlier work (C.F. Beaulieu, J.I. Clark, R.D. Brown III, M. Spiller, and S.H. Koenig, Magn. Reson. Med. 8, 45 (1988] on the magnetic field dependence of 1/T1 (NMRD profiles) of calf lens nuclear homogenates, at 25 degrees C, to 5 degrees C, and to other protein systems as well. These include concentrated solutions of myoglobin and bovine serum albumin, both globular proteins, the first compact and roughly spherical, the other extended, flexible, and with weak internal bonding; chicken lens homogenate, for which the dominant crystallins (lens proteins) are approximately 70% alpha-helical compared with calf crystallins, which are essentially all beta-sheet; and hen egg white, both native and heat-denatured. Our earlier conjectures regarding a reversible change in protein organization of the calf lens crystallins as a function of solute protein concentration is given added support. Our findings suggest that cytoplasmic homogenate can be characterized as a heterogeneous and polymorphic solution of crystallins. At high concentrations the NH moieties of the protein backbone become accessible to solvent with water (not NH proton) exchange rates greater than 10(4) s-1. This conclusion is based on two aspects of the observed NMRD profiles. At low crystallin concentration, the profiles of calf and chicken lens homogenates are similar in form to those of myoglobin and native hen egg white, a form that has been studied previously for a range of diamagnetic globular proteins and has been demonstrated to arise from the rotational thermal motion of the solute molecules. At high crystallin concentrations, the NMRD profiles of the lens homogenates develop a monotonic background (high rates at low fields), much like that of the heat-denatured egg-white sample and those of most tissues. In addition, there is a set of peaks in the central part of the profiles of the concentrated crystallins, seen also in the denatured egg white and some tissues but not in the myoglobin sample, which is known to arise from cross-relaxation interactions between the water protons and (through the intermediary of the NH proton) the 14N quadrupolar levels. The magnitude of these peaks, which is larger by an order of magnitude for native calf lens homogenates than for any tissue, requires that the majority of the NH moieties be accessible to water. Finally, going to 5 degrees C for the native calf lens homogenate takes the sample below the temperature of reversible phase separation, and it becomes opaque.(ABSTRACT TRUNCATED AT 400 WORDS)
我们将之前关于小牛晶状体核匀浆在25℃时1/T1的磁场依赖性（NMRD曲线）的研究工作扩展到了5℃，并扩展到了其他蛋白质体系。这些体系包括肌红蛋白和牛血清白蛋白的浓溶液，二者均为球状蛋白，前者结构紧密且大致呈球形，后者结构伸展、灵活且内部键合较弱；鸡晶状体匀浆，其主要晶状体蛋白（晶状体蛋白）约70%为α螺旋结构，而小牛晶状体蛋白基本上全是β折叠结构；还有生的和热变性的鸡蛋清。我们之前关于小牛晶状体蛋白的蛋白质组织随溶质蛋白浓度发生可逆变化的推测得到了更多支持。我们的研究结果表明，细胞质匀浆可被表征为晶状体蛋白的非均匀多晶型溶液。在高浓度下，蛋白质主链的NH基团可与溶剂接触，水（而非NH质子）的交换速率大于10⁴ s⁻¹。这一结论基于所观察到的NMRD曲线的两个方面。在低晶状体蛋白浓度下，小牛和鸡晶状体匀浆的曲线在形式上与肌红蛋白和生鸡蛋清的曲线相似，这种形式此前已针对一系列抗磁性球状蛋白进行过研究，并且已证明是由溶质分子的旋转热运动引起的。在高晶状体蛋白浓度下，晶状体匀浆的NMRD曲线会出现单调背景（低场处速率较高），这与热变性蛋清样品以及大多数组织的情况非常相似。此外，在浓晶状体蛋白曲线的中部有一组峰，在变性蛋清和一些组织中也能看到，但在肌红蛋白样品中未出现，已知这是由水质子与（通过NH质子的中介）¹⁴N四极能级之间的交叉弛豫相互作用引起的。这些峰的大小，对于天然小牛晶状体匀浆来说比任何组织都大一个数量级，这表明大多数NH基团可与水接触。最后，将天然小牛晶状体匀浆冷却到5℃会使样品低于可逆相分离的温度，并且它会变得不透明。（摘要截选至400字）
