Introduction:
The process of molecular self-assembly(SA) is central to all biological processes. In addition to exploring natural SA systems for their own sake, chemists, biochemists and physicists are increasingly turning to them for inspiration for applications in biotechnology and nanotechnology. We have used the coiled-coil motif as a building block for SA. Many natural coiled coils extend over hundreds of amino acids and assemble further to form thicker, multi-stranded filaments (e.g. intermediate filaments) (Herrmann & Aebi, 1998, Curr Opin Struct Biol 8, 177). Taking these structures as inspiration the lab has a designed a self-assembling system based on short peptides that form coiled coils.
Two 28-residue peptides, dubbed Self-Assembling Fibre peptides (SAF-p1 and SAF-p2), were designed to assemble into a long coiled-coil fibre when mixed. The design is based on the idea of a "sticky end" assembly rather than a blunt ended assembly, which is normally seen in natural coiled-coil systems.
Isoleucine at positions a, and leucine at positions d were included to select for a parallel coiled-coil dimer, whilst oppositely-charged residues at e and g were used to promote staggered heterodimer with complementary overhangs. Asparagine residues (*) introduced at different a positions. These preferentially pair with each other in the core (Gonzalez et al, 1996, Nature Struct Biol 3, 1011), to ensure the off-set register of the two peptides.
Characterization.
Circular dichroism (CD) Spectroscopy:
Spectra of the individual peptides at 100uM in aqueous solution show that the peptides are unfolded. However, spectra of mixtures of the two peptides show a distinctive alpha-helical signal, with a negative minima at 222nm and 208nm.
Microscopy.
We can use a variety of different microscopical techniques to visualise different aspects of the fibres and at different scales.
Electron Microscopy:
To visualise the fibres at high magnification we use electron microscopy. This reveals fibrils of approximately 70nm in width and many microns long. Since a coiled-coil has a width of 2nm, the fibres are built up from laterally assembled coiled-coils. We can control the morphology of the fibres by altering the conditions under which they are grown.
Light Microscopy
We can specifically label the peptides with different fluorophores and incorporate these into the fibres to produce labelled fibres. By adding fluoroscein- and rhodamine-labelled peptides at different times, it is possible to label specific parts of the fibres.
These fibres were formed by incubating the individual peptides in the presence of a peptide labelled with fluoroscein for 24 hours, then a small amount of peptide labelled with rhodamine was added after 24 hours. The second label specifically recruits to one end so, which shows that the fibres are polar.
Atomic Force Microscopy (AFM)
We can use the AFM to look at the morphology of the fibres after they have been dried down on a surface, or when they are immobilised on the surface under solution. This allows us to check that the visualistion techniques that we are using (for example the TEM) are not destroying the sample, or introducing artefacts from the imaging process. This also allows us to obtain accurate measurements of the heights of fibres.
200nm Height Scale
X-ray Fibre Diffraction:
In addition, we are using X-ray fibre diffraction to determine the coiled-coil assembly in orientated fibres. Initial results show a meridional arc at 5.13Å which is characteristic of coiled coils and an equatorial arc at 9.2Å,characteristic of the spacing between alpha-helices in coiled coils.
X-ray fibre diffraction pattern of mature fibres. These reflections imply coiled-coil structure. Data collected by M.Sunde at Cambridge University.
This work is further discussed in :
"Sticky-end Assembly of a Designed Peptide Fiber Provides Insight Into Protein Fibrillogenesis",
MJ Pandya et al., Biochemistry, 39 8727-8734, (2000) (Abstract).
"Polar assembly in a designed protein fiber",
AM Smith et al., Angew. Chem. Int. Ed. 44 325-328, (2005) (Abstract)