Laboratories of Biophysics and Nanotechnology, University of Genoa Medical School, Genoa, Italy.
Fondazione EL.B.A. - Nicolini, Pradalunga, Italy.
Nat Protoc. 2017 Dec;12(12):2570-2589. doi: 10.1038/nprot.2017.108. Epub 2017 Nov 30.
The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.
新一代的同步加速器和微聚焦光束线使得 X 射线蛋白质晶体学取得了巨大的进展，为制药行业和生命科学领域高度关注的蛋白质提供了新的三维原子结构。然而，通常仍然难以生产出具有足够大小和质量（有序性、衍射强度、辐射稳定性）的蛋白质晶体。在本方案中，我们提供了执行 Langmuir-Blodgett（LB）纳米模板方法的说明，这是一种可用于任何蛋白质（包括膜蛋白）的结晶方法。我们描述了如何在气液界面上制备高度有序的二维 LB 蛋白质单层，并将其沉积在载玻片上。可以通过表面压力测量和布鲁斯特角显微镜（BAM）观察 LB 膜的形成，尽管其质量可以通过原子力显微镜（AFM）和纳米重力测量来表征。然后，这些薄膜可以用作通过悬滴气相扩散触发三维蛋白质晶体形成的二维模板。形成二维模板的过程只需几分钟。使用原位亚微米 GISAXS（掠入射小角 X 射线散射）方法可以获得蛋白质在 LB 膜中结晶过程中的纳米级重组结构信息。根据本方案中描述的方法，可以直接在 LB 膜和蛋白质溶液的界面上实时测量微 GISAXS 光谱，并根据层、岛或孔的形成来解释。根据我们的经验，获得的 LB 晶体需要 1-10 天来准备，与使用其他结晶方法相比，它们具有更高的有序性和辐射稳定性。
