Martin Rachel W, Zilm Kurt W
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-8107, USA.
J Magn Reson. 2003 Nov;165(1):162-74. doi: 10.1016/s1090-7807(03)00253-2.
Preparation of proteins in their crystalline state has been found to be important in producing stable therapeutic protein formulations, cross-linked enzyme crystals for application in industrial processes, generating novel porous media for separations, and of course in structure elucidation. Of these applications only X-ray crystallography requires large crystals, defined here as being crystals 100s of microns or greater in size. Smaller crystals have attractive attributes in many instances, and are just as useful in structure determination by solid state NMR (ssNMR) as are large crystals. In this paper we outline a simple set of procedures for preparing nanocrystalline protein samples for ssNMR or other applications and describe the characterization of their crystallinity by ssNMR and X-ray powder diffraction. The approach is demonstrated in application to five different proteins: ubiquitin, lysozyme, ribonuclease A, streptavidin, and cytochrome c. In all instances the nanocrystals produced are found to be highly crystalline as judged by natural abundance 13C ssNMR and optical and electron microscopy. We show for ubiquitin that nanocrystals prepared by rapid batch crystallization yield equivalent 13C ssNMR spectra to those of larger X-ray diffraction quality crystals. Single crystal and powder X-ray diffraction measurements are made to compare the degree of order present in polycrystalline, nanocrystalline, and lyophilized ubiquitin. Solid state 13C NMR is also used to show that ubiquitin nanocrystals are thermally robust, giving no indication of loss of local order after repeated temperature cycling between liquid nitrogen and room temperature. The methods developed are rapid and should scale well from the tenths of milligram to multi-gram scales, and as such should find wide utility in the preparation of protein nanocrystals for applications in catalysis, separations, and especially in sample preparation for structural studies using ssNMR.
人们发现，制备处于结晶状态的蛋白质对于生产稳定的治疗性蛋白质制剂、用于工业过程的交联酶晶体、生成用于分离的新型多孔介质，当然还有结构解析都很重要。在这些应用中，只有X射线晶体学需要大晶体，这里将大晶体定义为尺寸在100微米或更大的晶体。在许多情况下，较小的晶体具有吸引人的特性，并且在通过固态核磁共振（ssNMR）进行结构测定时与大晶体一样有用。在本文中，我们概述了一套简单的程序，用于制备用于ssNMR或其他应用的纳米晶体蛋白质样品，并描述了通过ssNMR和X射线粉末衍射对其结晶度的表征。该方法通过应用于五种不同的蛋白质进行了演示：泛素、溶菌酶、核糖核酸酶A、链霉亲和素和细胞色素c。在所有情况下，通过天然丰度13C ssNMR以及光学和电子显微镜判断，所产生的纳米晶体都具有高度结晶性。我们表明，对于泛素，通过快速批量结晶制备的纳米晶体产生的13C ssNMR光谱与较大的X射线衍射质量晶体的光谱相当。进行了单晶和粉末X射线衍射测量，以比较多晶、纳米晶和冻干泛素中存在的有序程度。固态13C NMR还用于表明泛素纳米晶体具有热稳定性，在液氮和室温之间反复温度循环后，没有显示出局部有序性丧失的迹象。所开发的方法快速，并且应该能够很好地从十分之一毫克扩展到多克规模，因此在制备用于催化、分离的蛋白质纳米晶体，特别是在使用ssNMR进行结构研究的样品制备中应该有广泛的用途。
