Structural Biochemistry/Membrane Proteins/Methods for Studying Membrane Proteins/Electron crystallography of proteins in membranes

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Electron and x-ray crystallography study of membrane proteins have confirmed the ‘fluid mosaic model’ of the membrane structure, proposed by Singer and Nicholson. The ‘fluid mosaic model’ pictured protein molecules integrated in the fluid lipid bilayer as isolated units or complex. To further understand the function of the membrane protein requires the study of their conformational changes in different physiological settings. Electron crystallography of protein – lipid arrays has played a key role in understanding of how membrane proteins work. In 1975, electron crystallography provided the first view of polypeptide chain of alpha-helices of bacteriorhodopsin traversing the lipid bilayer. Since 1975, electron crystallography has been able to unveil other structures and how polypeptide folds. Electron crystallography is able to study membrane proteins in trapped transition state, and samples frozen rapidly in a defined solution. New methodological advances have also been made allowing the collection of detailed information of small or disordered membrane assemblies.

Sheet and Tubes[edit]

Figure 1: Two types of protein crystal used for electro crystallography. Left one is proteins frozen in sheet like 2D and right one is tubular. Molecules arranged on the surface of tubular crystal have helical symmetry, giving the rise to many different of views.

Electro crystallography is the study of proteins in crystallized form which reveals their 3D structure and amino acid sequence. There are two types of crystals made for studies on protein-lipid arrays (Figure 1). One is in the form of sheets (2D) and the other in the form of tubes (tubular shape). The lattice and symmetry of the two forms define accurately the position and orientation of each molecule. Using the advantage of these two types, the crystals have revealed precise details of individual lipid molecules and protein side chains.

Tubular crystals resemble the natural product of protein crystallizing on the surface of vesicles, but they have not been used extensively for determining exact 3D structure because it requires relatively large proteins (>250 kDa) to obtain accurate 3D shape averaging the isolated proteins.

Figure 2: Portion of tobacco mosaic virus and fitted polypeptide chain using real-space reconstruction method.

Traditionally, the Fourier-Bessel methods are used to determine the 3D structure of a protein. The image of the crystal is processed to correct for distortion. The repeat length is divided into short segment and compared to a reference structure to determine the parameters of the 3D alignment. An alternative real-space method is now widely used treating segments as a string of single particles. This method has been applied to tubular protein-lipid crystals (Figure 2), demonstrating its potential to determine their structure at near-atomic resolution.

Freeze-trapping different conformational states[edit]

The principle value in using tubular and sheet crystals is that they probe the structures of membrane proteins in their natural lipid environment; therefore other methods carry the risk of not representing a biological relevant state. To capture protein-in-lipid specimens, a routine step is plunge-freezing of the electron microscope grid into liquid nitrogen-cooled ethane. The low temperature cools the protein specimens very rapidly and trapping them in their transient structure, which has a life-time of a millisecond or longer.

The freeze-trapping technique allowed a structural model of the membrane gating mechanism to be proposed but high quality images of ACh receptor channel in tubular crystals have been difficult to obtain. Recently, the development of an extremely stable helium-cooled top-entry stage has made the entire process of data collection much easier, and should eventually permit the gating mechanism to be described at near-atomic resolution.

Molecular Tomography[edit]

Cryo-electron tomography is a recently developed approach to explore membrane protein in their functional state. With this technique, the 3D picture of frozen specimen is built up by combining information from a large number of tilt views. The organization of proteins can be obtained from the tomographic section. The atomic structures obtained from other methods can be docked into the tomographic densities to show proteins in their proper functional context. Recent tomographic study of frozen sections highlights the potential for exploring the architecture of more complex membrane assemblies and organelles.

Special properties of proteins in membranes[edit]

Figure 3: Domain structures of ligand-gated ion channel and an ABC transporter. Between the upper ligand binding and the middle transmembrane domain exist an interface that is involved in coupling mechanically distinct conformational changes. The upper ligand binding is a control element to switch configuration of the helices in the membrane section. The interface itself is in black.

From electron and x-ray crystallographic studies, the right-handed helix packing is common in membrane protein and more stable in bilayer environment. Helices in membrane also adopt bent configurations, which enable them to pack more tightly against each other. Helices may contain flexible region or hinge point, as in bacteriorhodopsin, to enable a rapid localized conformation change.

Many membrane proteins do not in fact undergo rigid quaternary rearrangement typical of other allosteric proteins, but instead work as a result of distinct movements in each domain, which are coupled to one another. Examples are ligand-gated ion channels and ABC transporters (figure 3).

Figure 4: Left is a KcsA potassium ion channel, which has a selectivity filter with carbonyl group lining the surface. The carbonyl groups act as substitute ideally for normally tightly bound water molecules allowing ion conduction across the membrane. Right is transmembrane portion of an ACh receptor. The gate is in fact much wider but the completely hydrophobic gate effective creates a permeation barrier to ions. Ions has no opportunity shed their hydration shells and so are too large to go through.

Ion channels in charge of ion flows across membrane share similar principles with soluble enzymes. Ion channels, like an enzyme can incorporate precise stereochemistry changes to bind to specific substrate and catalyze reactions. The potassium channel uses precisely positioned carbonyl group lining its surface to provide a constricting, yet highly conductive pathway for potassium ions across a part of bilayer (figure 4). By contrast, the closed-gate in ACh receptor has a much wider opening but is built of completely hydrophobic residues. This creates an effective permeation barrier to potassium ions, because ions has no opportunity to drop its hydration shell and therefore too large to pass through.

Conclusion[edit]

The structural and chemical information about membrane proteins have been acquired from images formed by electron crystallography combined with advances in new techniques such as in cryo-technology. Compared to X-ray diffraction of proteins in detergent, these methods preserve the biology relevance of the structure. The development of tomography and real-space averaging methods are extending the possibilities of obtaining high resolution information from increasingly smaller protein-lipid arrays. Improvements in methods and ultimately the development of better electron detectors are keys to future research on membrane proteins.[1]

References[edit]

  1. Yoshinori Fujiyoshi1 and Nigel Unwin2, 1Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. 2MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. Link text, additional text.