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New set of standards allows accurate protein counting
August 2015. Large protein assemblies (>20 nm in diameter), such as nuclear pores, transcription factories or centrioles, are not accessible to structure determination by X-ray crystallography and are less well understood as a result. In the case of the nuclear pore, cutting-edge experiments have revealed many structural details but estimates of its mass and subunit stoichiometry differed by factors of two or more.
One way to investigate the structural details of large protein assemblies is to determine the copy number of tagged subunits by using fluorescence microscopy. This can be achieved by analyzing step-wise photobleaching or by comparing the total intensity of a structure to those of standards containing known numbers of tags. The combination of such counting strategies with super-resolution techniques that are able to sensitively resolve individual protein complexes holds immense promise.
A general problem that reduced the robustness of all microscopy-based counting methods was that no reliable and easy-to-use standards were available to test their accuracy. A team of Frankfurt scientists led by Mike Heilemann has developed a selection of bacterial homo-oligomers for easy validation and calibration of molecular counting methods. Their homo-oligomers contain 10–24 subunits and fully assemble when expressed in mammalian cells, as the scientists report in the journal Angewandte Chemie. The usefulness of these standards was demonstrated by the team being able to establish that nuclear pores contain 32 copies of the Nup107 complex.
The new standards can be used to convert the intensity scales of fluorescence microscopy images into molecular units through a simple method that can be used with any microscope capable of producing z-stacks. DNA encoding three out of the four homo-oligomers is available in many labs and the ability to check the relative intensities of multiple standards provides a powerful internal control. The homo-oligomers are expected to be compatible with many types of fluorescence microscopy, including SIM and STED. These standards will also help to improve existing localization-microscopy-based counting methods by aiding users in determining molecular detection efficiencies and by serving as positive controls to help assure nonspecialists that the complex corrections associated with such techniques have functioned correctly. More ...
Contact:
Mike Heilemann, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max von Laue Straße 9, 60438 Frankfurt, Germany, Heilemann@chemie.uni-frankfurt.de
Full reference:
Finan K, Raulf A, Heilemann M (2015) A set of homo-oligomeric standards allows accurate protein counting. Angew Chem Int Ed, Epub ahead of print 20 August 2015, doi: 10.1002/anie.201505664. Link