High-throughput methods for electron crystallography.

Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA, .
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2013; 955:273-96. DOI: 10.1007/978-1-62703-176-9_15
Source: PubMed

ABSTRACT Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

  • [Show abstract] [Hide abstract]
    ABSTRACT: For small helical membrane proteins their structure is highly sensitive to their environment and solid state NMR is a structural technique that can characterize these membrane proteins in native like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation. A membrane protein from M. tuberculosis is used to illustrate the method. The results show that a detergent that stabilizes the most protein is not always ideal and sometimes cannot be removed by dialysis. By focusing on the lipid and protein binding properties of the detergent, proteoliposome preparations can be readily produced which provide double signal the to noise ratios for both oriented sample and magic angle spinning solid state NMR. The method will allow more membrane protein drug targets to be structurally characterized in lipid bilayer environments.
    Biochemistry 03/2014; 53(15). DOI:10.1021/bi500144h · 3.01 Impact Factor