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Langmuir–Blodgett nanotemplates for protein crystallography

Nature Protocols volume 12, pages 2570–2589 (2017)Cite this article

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Abstract

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.

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Figure 1: LB protein nanotemplate.
Figure 2: Characterization of protein LB thin films.
Figure 3: Representation of the principle of μGISAXS measurement.
Figure 4: Bovine cytochrome P450scc data.
Figure 5: Human protein kinase CK2-α data.
Figure 6: Schematic view of in situ microGISAXS flow crystallization cell.
Figure 7: Actual experimental setup of in situ submicron GISAXS, as described in ref. 34.
Figure 8: Set of photographs for Langmuir–Schaefer preparation of a protein nanotemplate on the cover glass slide.
Figure 9: Examples of crystals formation by classic and LB nanotemplate methods.
Figure 10: Typical microGISAXS patterns of LB versus classic crystal formation.
Figure 11: A vertical cut through the 2D microGISAXS pattern is shown to demonstrate the time evolution of the Yoneda peak formation for a lysozyme crystal.

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Acknowledgements

This work was supported by a FIRB RBPR05JH2P Nanoitalnet grant on Organic and Biological Nanosensors, financed by the Italian Ministry of Education, Universities and Research (MIUR) to C.N. at the University of Genoa (co-principal investigator E.P.); an International FIRB-MIUR RBIN04RXHS grant together with Harvard University on Functional Proteomics and Cell Cycle Progression to C.N. at the University of Genoa (co-principal investigator E.P.); a FIRB RBNE01X3CE grant on Organic Nanotechnologies and Nanosciences financed by MIUR to Fondazione EL.B.A.; (principal investigator E.P.); a PNR-MIUR grant on Biocatalysis to C.N. at the University of Genoa and Fondazione EL.B.A.; and a yearly MIUR Grant for Functioning to Fondazione EL.B.A.; – Nicolini. The authors are grateful to C. Riekel (ESRF) for a long-lasting collaboration in the microGISAXS experiments.

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Authors and Affiliations

  1. Laboratories of Biophysics and Nanotechnology, University of Genoa Medical School, Genoa, Italy

    Eugenia Pechkova & Claudio Nicolini

  2. Fondazione EL.B.A. - Nicolini, Pradalunga, Italy

    Eugenia Pechkova & Claudio Nicolini

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  1. Eugenia Pechkova
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Contributions

E.P. and C.N. contributed equally to this work. E.P. performed LB-film depositions and crystallization experiments under the supervision of C.N., E.P. prepared samples for synchrotron X-ray crystallography and microGISAXS. The LB nanotemplate-based flow crystallization cell for in situ microGISAXS experiments was designed by E.P. Both authors collected the data, and C.N. performed microGISAXS data analysis. The manuscript was prepared by E.P., with the discussion of LB method state of the art written by C.N.

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Correspondence to Eugenia Pechkova.

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Supplementary information

Supplementary Methods

Cloning, expression and purification of target-membrane protein cytochrome P450scc. (PDF 135 kb)

Langmuir–Blodgett (LB) nanotemplate deposition procedure.

Protein monolayer transfer is achieved by touching the monolayer with the glass slide in parallel to the air–water interface of the LB trough, followed by horizontal lift according to the Langmuir–Schaefer (LS) technique. The slide can be held by forceps or by a cover slide vacuum gadget (Hampton Research, cat. no. HR8-098). (MOV 2826 kb)

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Pechkova, E., Nicolini, C. Langmuir–Blodgett nanotemplates for protein crystallography. Nat Protoc 12, 2570–2589 (2017). https://doi.org/10.1038/nprot.2017.108

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