Novel nanotechnology approach to help visualise proteins

A team of Oxford researchers has developed a patent-pending platform technology to aid single-molecule visualisation techniques that are used to determine protein structure.

DNA-templated protein arrays. On the left, a diagram of the self-assembled DNA template ('DNA template') is shown superimposed on a projected density map obtained by cryo-EM. The template may be modified (*) to present clusters of binding sites for a protein arranged on a hexagonal lattice. On the right, a cryo-electron micrograph of soluble G-protein G i1, bound to a trisNTA-functionalised template through a (His)6 affinity tag is shown ('Protein array'). Reprinted with permission from Nano Letters 11 (2), 657-660. Copyright 2011 American Chemical Society

DNA-templated protein arrays. Reprinted with permission from Nano Letters 11 (2), 657-660. Copyright 2011 American Chemical Society. On the left, a diagram of the self-assembled DNA template (‘DNA template’) is shown (more...)

The researchers, based in the Departments of Biochemistry and Physics, and in the Division of Structural Biology, expect the approach to play an important part in enabling the wide application of these techniques, in particular to membrane proteins which form the largest class of drug targets. The groups, led by Robert Gilbert, Anthony Watts and Andrew Turberfield, have published their work in Nano Letters 1.

Determining the structure of target proteins is a major bottleneck in pre-clinical drug discovery. Researchers traditionally use the technique of X-ray crystallography. But for proteins associated with biological membranes, this technique is hampered because these proteins can be difficult to express, solubilise and crystallise.

With recent advances, a complementary imaging technique - electron microscopy - has emerged. Single-particle cryo-electron microscopy (cryo-EM) allows direct visualisation of biomolecules, flash-frozen in a solution suspended across holes in a carbon film. This rapidly developing technique permits structural characterisation of non-crystalline protein samples and is therefore particularly promising for hard-to-crystallise membrane proteins and protein complexes. However, its use is severely limited by the extremely poor signal-to-noise ratio and the requirement to collect and process vast amounts of data.

The researchers have developed a technique that dramatically increases the throughput of data collection. It relies on self-assembled DNA nano-affinity templates to produce dense protein arrays that are optimized for structure determination by cryo-EM (see figure).

Using these templates, they have demonstrated that the technique can be applied to samples that span the range of potential targets for single-particle cryo-EM - a soluble G-protein, a G-protein coupled membrane receptor, and a functional complex of the two.

This is a crucial time for the introduction of this technique; successful application of high-resolution single-particle cryo-EM will require the acquisition of tens of thousands or millions of particle images. DNA nano-affinity templates allow this scale of data collection from a single micrograph, and they are cheap and easy to implement. This new development therefore looks likely to address a looming bottleneck in the field.

Reference

  1. Selmi, D. N. et al. (2011). DNA-Templated Protein Arrays for Single-Molecule Imaging. Nano Lett. 11 (2): 657-660.

 





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