Membrane Proteins Structural Determination

Membrane proteins are crucial components in cellular physiology and biochemistry. Membrane proteins are also involved in processes of disease, and they are the molecular targets of over 40% of all FDA-approved drugs. Determination of the atomic-level structure of membrane proteins will go a long way toward understanding how these protein molecules affect biochemical and physiological processes. However, structure determination of membrane proteins is often considered as greatest challenges. Until now, structural information on membrane proteins is sparse. Thus, rational approaches to overcoming the bottlenecks in the field are urgently required. Alfa Chemistry provides high quality structural determination service to scientists and researchers who need to solve the precise structure of membrane proteins to facilitate their research, development, and applications of membrane proteins.

Structural Determination Technologies

Alfa Chemistry offers three types of structure determination methods for your membrane proteins, including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM).

X-ray Crystallography

The predominate technique in membrane protein structural biology had been X-ray crystallography — indeed, most of the membrane protein structures in the protein databank were determined by X-ray crystallography. Alfa Chemistry provides X-ray crystallography services for accurate membrane protein structure determination with our state-of-the-art equipment and rich experience. It is worth mentioning that high quality membrane protein crystal is the basis of this technique. So, how to obtain high quality crystal is one of the key links to study the structure of membrane protein by X-ray crystallography. Our company uses a variety of techniques to promote diffraction quality of crystals. These techniques are respectively based on detergent (In surfo method) and lipid (In cubo method) systems.

• In surfo method: The basis of the in surfo method is the incorporation of protein (and any residual lipid that may be present) into detergent micelles. The production of crystals can then occur via standard routes such as vapour diffusion or microdialysis. To date, this approach has been successfully employed in the crystallization of cytochrome c oxidase, cytochrome b1c and the KcsA potassium channel.
• In cubo method: The in cubo method of crystallization works on the basis that a membrane protein should crystallize with greater ease in an environment of lipid which is similar to its natural bilayer. Therein, lipid cubic phase (LCP) and lipid sponge phase (LSP) has already solved countless tertiary structures of membrane proteins. These two technologies are our featured services. 

Fig. 1. Schematic structure of LCP and LSP.

NMR Spectroscopy

NMR spectroscopy is becoming one of the main methods to solve the structure of membrane proteins because it does not need to prepare the crystal sample of membrane proteins and can obtain the three-dimensional structure information of proteins with high resolution. In addition, different NMR methods can be used to study the three-dimensional structure of membrane proteins in different environments, as well as the dynamic properties of membrane proteins at different time scales. The NMR methods used by Alfa Chemistry to study the structure of membrane proteins mainly include solution and solid-state NMR spectroscopy. Thereinto, the solution NMR method is mainly used in membrane protein/detergent micelle complex, while solid-state NMR is applied to membrane protein/phospholipid complexes.

Fig. 2. Dynamic structure of the OmpA TM domain. The structure was solved by solution NMR. Backbone dynamics were probed by heteronuclear NOE measurements of all colored residues and mapped onto the structure using a color code ranging from blue (most rigid) to red (most dynamic). ND, not determined ^[1].

cryo-EM

Multiple results have demonstrated the accuracy of cryo-EM for protein structure determination, which allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. However, biomolecules are beam sensitive and produce a low image contrast in cryo-EM technology, extensive image averaging is required to retrieve high-resolution structural information. Image averaging can most efficiently be carried out when the molecules are packed in a 2D crystal. Alfa Chemistry adopts four main techniques that are used for 2D crystallization: (i) the Biobead method (ii) the dialysis method; (iii) the dilution method (the lipid-detergent-protein mixture is diluted to bring the detergent below the critical-micellar concentration (CMC)); and (iv) the monolayer method.

Fig. 3. Flow diagram of preparation of membrane protein 2D crystals.

Order Process

Reference

1. Arora A., et al. Biophysical approaches to membrane protein structure determination[J]. Current opinion in structural biology, 2001, 11(5): 540-547.

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